Transplant media

ABSTRACT

The present invention relates to media containing purified antimicrobial polypeptides, such as defensins, and/or cell surface receptor binding proteins. The media may also contain buffers, macromolecular oncotic agents, energy sources, impermeant anions, ATP substrates. The media find use for the storage and preservation of internal organs prior to transplant.

This application is a continuation of U.S. application Ser. No.09/917,340, filed Jul. 27, 2001, now issued as U.S. Pat. No. 6,696,238,which claims priority to U.S. provisional applications 60/221,632, filedJul. 28, 2000, 60/249,602, filed Nov. 17, 2000, and 60/290,932, filedMay 15, 2001.

FIELD OF THE INVENTION

The present invention relates to media comprising purified antimicrobialpeptides, pore forming agents, and/or cell surface receptor bindingcompounds and their use for the storage and preservation of organs priorto transplant.

BACKGROUND OF THE INVENTION

A wide variety of organs, including kidneys, lungs, livers, hearts,pancreases, and small intestines are routinely and successfullytransplanted. These organs are obtained either from living donors orfrom cadaveric sources.

In 1998, a total of 12,166 kidney transplants were performed in theUnited States by programs tracked by the UNOS Transplant PatientDataSource. A total of 45,189 people were on the waiting lists forkidneys as of Sep. 30, 1999. Over 20,000 kidneys were transplantedbetween Jul. 1, 1995 and Jun. 30, 1997. The graft survival rate forthese transplanted kidneys was 93.4% after three months.

The ability to store organs for two or three days prior totransplantation allows sufficient time for histo-compatibility testingof donor and recipient, transport of the organ between transplantcenters, preoperative preparation of the recipient, preliminary donorculture testing, and vascular repair of the organ if needed. Theefficacy of organ transplantation depends in part on how well the organis preserved prior to transplantation. Two methods are used to preserveorgans prior to transplant: hypothermic storage and continuous pulsatileperfusion. Hypothermic storage by simple cold storage methods involvesremoval of an organ from a donor followed by rapid cooling. Cooling isachieved by a combination of external cooling and a short period ofperfusion with a chilled medium to reduce the core temperature of theorgan as quickly as possible. The organs are then immersed in aflush-out medium at from 0° C. to 4° C. Continuous pulsatile perfusioninvolves the continuous infusion of organs with a preservation solutiondesigned to prevent low temperature injury.

A number of media have been developed for infusing and preserving organsprior to transplantation. Examples of such media include VIASPAN (alsoknown as University of Wisconsin solution; Barr Laboratories, Pomona,N.Y.), University of Wisconsin Machine Perfusion Solution, HypertonicCitrate Solution, histindine-tryptophan-glutarate solution (HTKSolution), HTK Solution of Bretschneider, Phosphate Buffered Sucrose,EuroCollins Solution, and Collins C2 Solution. However, none of thesemedia are able to extend the preservation of organs past about 72 hoursusing cold storage methods. Additional preservation time would be usefulfor tests and for transportation of the organs. Furthermore, media thatincrease preservation time also can be expected to provide healthierorgans for transplants performed within 72 hours.

Accordingly, what is needed in the art are improved media for preservingand storing organs prior to transplant. Such media should be able toextend the preservation period past 72 hours and provide organs withincreased functionality upon transplant.

SUMMARY OF THE INVENTION

The present invention relates to media comprising antimicrobialpolypeptides or pore forming agents and/or cell surface receptor bindingcompounds and their use for the storage and preservation of organs priorto transplant.

The present invention is not limited to any particular media orformulation. Indeed, a variety of medias and formulations arecontemplated. In some embodiments, the present invention providescompositions comprising a purified antimicrobial polypeptide andhydroxyethyl starch. The present invention is not limited to anyparticular antimicrobial peptide. Indeed a variety of antimicrobialpeptides are contemplated, including, but not limited to, thoseidentified by SEQ ID NOs:1-96. In some preferred embodiments, theantimicrobial peptide is a defensin. The present invention is notlimited to any particular defensin. Indeed, the use of a variety ofdefensins is contemplated, including, but not limited to thoseidentified by SEQ ID NOs:37-96. In particularly preferred embodiments,the antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO:37). In some preferred embodiments, the antimicrobial polypeptide ordefensin comprises D-amino acids. In some embodiments, the antimicrobialpeptide and hydroxyethyl starch are in solution. The media of thepresent invention are not limited to any particular concentration ofantimicrobial peptide. Indeed, a range of concentrations arecontemplated (e.g., from about 0.01 to 1000 mg/l and preferably fromabout 0.1 to 5 mg/l). The present invention is not limited to anyparticular concentration of hydroxyethyl starch. Indeed, a range ofconcentrations are contemplated (e.g., from about 1 to 200 g/l). In someembodiments, the media further comprises a cell surface receptor bindingcompound. The present invention is not limited to any particular cellsurface receptor binding compound. Indeed, a variety of cell surfacereceptor binding compounds are contemplated, including, but not limitedto insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF),nerve growth factor (NGF), and substance P.

In other embodiments, the present invention provides compositionscomprising an antimicrobial polypeptide and an impermeant anion selectedfrom the group consisting of lactobionic acid and gluconate. In somepreferred embodiments, the antimicrobial polypeptide and the impermeantion are in solution. The present invention is not limited to anyparticular antimicrobial peptide. Indeed a variety of antimicrobialpeptides are contemplated, including, but not limited to, thoseidentified by SEQ ID NOs:1-96. In some preferred embodiments, theantimicrobial peptide is a defensin. The present invention is notlimited to any particular defensin. Indeed, the use of a variety ofdefensins is contemplated, including, but not limited to thoseidentified by SEQ ID NOs:37-96. In some preferred embodiments, theantimicrobial polypeptide or defensin comprises D-amino acids. Inparticularly preferred embodiments, the antimicrobial peptide is bovinedodecapeptide or BNP-1 (SEQ ID NO: 37). The media of the presentinvention are not limited to any particular concentration ofantimicrobial peptide. Indeed, a range of concentrations arecontemplated (e.g., from about 0.01 to 1000 mg/l and preferably fromabout 0.1 to 5 mg/l). The media of the present invention are not limitedto any particular concentration of impermeant ion. Indeed, a range ofconcentrations are contemplated (e.g., from about 1 to 500 mM). In someembodiments, the media further comprises a cell surface receptor bindingcompound. The present invention is not limited to any particular cellsurface receptor binding compound. Indeed, a variety of cell surfacereceptor binding compounds are contemplated, including, but not limitedto IGF-1, EGF, NGF, and substance P. In some preferred embodiments, themedia does not require the use of hydroxyethyl starch.

In other embodiments, the present invention provides compositionscomprising an antimicrobial polypeptide and glutathione. In somepreferred embodiments, the antimicrobial polypeptide and the impermeantion are in solution. The present invention is not limited to anyparticular antimicrobial peptide. Indeed a variety of antimicrobialpeptides are contemplated, including, but not limited to, thoseidentified by SEQ ID NOs:1-96. In some preferred embodiments, theantimicrobial peptide is a defensin. The present invention is notlimited to any particular defensin. Indeed, the use of a variety ofdefensins is contemplated, including, but not limited to thoseidentified by SEQ ID NOs:37-96. In some preferred embodiments, theantimicrobial polypeptide or defensin comprises D-amino acids. Inparticularly preferred embodiments, the antimicrobial peptide is bovinedodecapeptide or BNP-1 (SEQ ID NO: 37). The media of the presentinvention are not limited to any particular concentration ofantimicrobial peptide. Indeed, a range of concentrations arecontemplated (e.g., from about 0.01 to 1000 mg/l and preferably fromabout 0.1 to 5 mg/l). The media of the present invention are not limitedto any particular concentration of glutathione. Indeed, a range ofconcentrations are contemplated (e.g., from about 0.1 to 100 mM). Insome embodiments, the media further comprises a cell surface receptorbinding compound. The present invention is not limited to any particularcell surface receptor binding compound. Indeed, a variety of cellsurface receptor binding compounds are contemplated, including, but notlimited to IGF-1, EGF, NGF, and substance P. In some preferredembodiments, the media does not require the use of hydroxyethyl starch.

In further embodiments, the present invention provides compositionscomprising a purified antimicrobial polypeptide and an ex vivo internalorgan. The present invention is not limited to any particular internalorgan. Indeed, a variety of internal organs are contemplated, including,but not limited to kidneys, hearts, lungs, small intestines, largeintestines, livers, and pancreases. The present invention is not limitedto organs from any particular species of animal. Indeed, use of organsfrom a variety of animals is contemplated, including organs from humans,pigs, and dogs. The present invention is not limited to any particularantimicrobial peptide. Indeed a variety of antimicrobial peptides arecontemplated, including, but not limited to, those identified by SEQ IDNOs:1-96. In some preferred embodiments, the antimicrobial peptide is adefensin. The present invention is not limited to any particulardefensin. Indeed, the use of a variety of defensins is contemplated,including, but not limited to those identified by SEQ ID NOs:37-96. Inparticularly preferred embodiments, the antimicrobial peptide is bovinedodecapeptide or BNP-1 (SEQ ID NO: 37). In some preferred embodiments,the antimicrobial polypeptide or defensin comprises D-amino acids. Themedia of the present invention are not limited to any particularconcentration of antimicrobial peptide. Indeed, a range ofconcentrations are contemplated (e.g., from about 0.01 to 1000 mg/l andpreferably from about 0.1 to 5 mg/l). In some embodiments, thecompositions further comprise a macromolecular oncotic agent. Thepresent invention is not limited to any particular macromolecularoncotic agent. Indeed, a variety of macromolecular oncotic agents arecontemplated, including, but not limited to hydroxyethyl starch,dextran, and glucose. In other embodiments, the composition furthercomprises an impermeant anion. The present invention is not limited toany particular impermeant anion. Indeed, a variety of impermeant anionsare contemplated, including, but not limited to, gluconate andlactobionic acid. In still further embodiments, the compositionscomprise glutathione. In some embodiments, the compositions furthercomprise a cell surface receptor binding compound. The present inventionis not limited to any particular cell surface receptor binding compound.Indeed, a variety of cell surface receptor binding compounds arecontemplated, including, but not limited to IGF-1, EGF, NGF, andsubstance P. In some preferred embodiments, the media does not requirethe use of hydroxyethyl starch.

In still other embodiments, the present invention provides methodscomprising a) providing cellular material and a solution comprising apurified antimicrobial polypeptide and b) storing the cellular materialin said solution comprising a purified antimicrobial peptide. Thepresent invention is not limited to the storage of any particularcellular material. Indeed, a variety of cellular materials arecontemplated, including but not limited to internal organs, skin, andgametes. In some preferred embodiments, the cellular material is aninternal organ. The present invention is not limited to any particularinternal organ. Indeed, a variety of internal organs are contemplated,including, but not limited to kidneys, hearts, lungs, small intestines,large intestines, livers, and pancreases. The present invention is notlimited to organs from any particular species of animal. Indeed, use oforgans from a variety of animals is contemplated, including organs fromhumans, pigs, and dogs. In some embodiments, the internal organ isinfused with the solution. The present invention is not limited to anyparticular antimicrobial peptide. Indeed a variety of antimicrobialpeptides are contemplated, including, but not limited to, thoseidentified by SEQ ID NOs:1-96. In some preferred embodiments, theantimicrobial peptide is a defensin. The present invention is notlimited to any particular defensin. Indeed, the use of a variety ofdefensins is contemplated, including, but not limited to thoseidentified by SEQ ID NOs:37-96. In particularly preferred embodiments,the antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO:37). In some preferred embodiments, the antimicrobial polypeptide ordefensin comprises D-amino acids. The media of the present invention arenot limited to any particular concentration of antimicrobial peptide.Indeed, a range of concentrations are contemplated (e.g., from about0.01 to 1000 mg/l and preferably from about 0.1 to 5 mg/l). In someembodiments, the compositions further comprise a macromolecular oncoticagent. The present invention is not limited to any particularmacromolecular oncotic agent. Indeed, a variety of macromolecularoncotic agents are contemplated, including, but not limited tohydroxyethyl starch, dextran, and glucose. In other embodiments, thecomposition further comprises an impermeant anion. The present inventionis not limited to any particular impermeant anion. Indeed, a variety ofimpermeant anions are contemplated, including, but not limited to,gluconate and lactobionic acid. In still further embodiments, thecompositions comprise glutathione. In some embodiments, the compositionsfurther comprise a cell surface receptor binding compound. The presentinvention is not limited to any particular cell surface receptor bindingcompound. Indeed, a variety of cell surface receptor binding compoundsare contemplated, including, but not limited to IGF-1, EGF, NGF, andsubstance P. In some preferred embodiments, the media does not requirethe use of hydroxyethyl starch.

In still further embodiments, the present invention providescompositions comprising a cell surface receptor binding compound andhydroxyethyl starch. The present invention is not limited to anyparticular cell surface receptor binding compound. Indeed, a variety ofcell surface receptor binding compounds are contemplated, including, butnot limited to IGF-1, EGF, NGF, and substance P.

In other embodiments, the present invention provides compositionscomprising a cell surface receptor binding compound and an internalorgan. In some embodiments, the compositions further comprise amacromolecular oncotic agent. The present invention is not limited toany particular macromolecular oncotic agent. Indeed, a variety ofmacromolecular oncotic agents are contemplated, including, but notlimited to hydroxyethyl starch, dextran, and glucose. In otherembodiments, the composition further comprises an impermeant anion. Thepresent invention is not limited to any particular impermeant anion.Indeed, a variety of impermeant anions are contemplated, including, butnot limited to, gluconate and lactobionic acid. In still furtherembodiments, the compositions comprise glutathione. In some preferredembodiments, the media does not require the use of hydroxyethyl starch.

In some embodiments, the present invention provides compositionscomprising trehalose and hydroxyethyl starch. In some preferredembodiments, the trehalose and hydroxyethyl starch are in solution. Thepresent invention is not limited to any particular concentration oftrehalose. Indeed, a range of concentrations are contemplated (e.g.,from about 1 mM to 30 mM). In some embodiments, the compositions furthercomprise an antimicrobial peptide and/or cell surface receptor bindingcompound. In some embodiments, the compositions further comprise a cellsurface receptor binding compound. The present invention is not limitedto any particular cell surface receptor binding compound. Indeed, avariety of cell surface receptor binding compounds are contemplated,including, but not limited to IGF-1, EGF, NGF, and substance P. Thepresent invention is not limited to any particular antimicrobialpeptide. Indeed a variety of antimicrobial peptides are contemplated,including, but not limited to, those identified by SEQ ID NOs:1-96. Insome preferred embodiments, the antimicrobial peptide is a defensin. Thepresent invention is not limited to any particular defensin. Indeed, theuse of a variety of defensins is contemplated, including, but notlimited to those identified by SEQ ID NOs:37-96. In particularlypreferred embodiments, the antimicrobial peptide is bovine dodecapeptideor BNP-1 (SEQ ID NO: 37). The media of the present invention are notlimited to any particular concentration of antimicrobial peptide.Indeed, a range of concentrations are contemplated (e.g., from about0.01 to 1000 mg/l and preferably from about 0.1 to 5 mg/l). In someembodiments, the compositions further comprise a macromolecular oncoticagent. The present invention is not limited to any particularmacromolecular oncotic agent. Indeed, a variety of macromolecularoncotic agents are contemplated, including, but not limited tohydroxyethyl starch, dextran, and glucose. In other embodiments, thecomposition further comprises an impermeant anion. The present inventionis not limited to any particular impermeant anion. Indeed, a variety ofimpermeant anions are contemplated, including, but not limited to,gluconate and lactobionic acid. In still further embodiments, thecompositions comprise glutathione.

In other embodiments, the present invention provides a kit comprising avessel containing a solution comprising a compound selected from thegroup consisting of lactobionate and hydroxyethyl starch; and a vesselcontaining an antimicrobial polypeptide. In some embodiments, theantimicrobial polypeptide is BNP-1. In other embodiments, the vesselcontaining an antimicrobial polypeptide further comprises a cell surfacereceptor binding compound. In further embodiments, the cell surfacereceptor binding compound is selected from the group consisting ofIGF-1, EGF, NGF, and substance P. In some embodiments, the kit furthercomprises instructions for combining said solution and the antimicrobialpolypeptide.

In still further embodiments, the present invention provides processesfor producing a storage solution comprising providing a solutioncomprising a compound selected from the group consisting of hydroxyethylstarch and lactobionate and a purified antimicrobial polypeptide; andcombining said solution with the purified antimicrobial polypeptide. Insome embodiments, the method further comprising the steps of providingat least one cell surface receptor binding compound and combining the atleast one cell surface receptor binding compound with the solution andthe antimicrobial polypeptide.

In some preferred embodiments, the present invention provides acomposition comprising hydroxyethyl starch or lactobionate and anantimicrobial polypeptide for use as an organ storage or perfusionsolution. In some embodiments, the composition further comprising a cellsurface receptor binding compound. In other preferred embodiments, thepresent invention provides a composition comprising a purifiedantimicrobial polypeptide (e.g., BNP-1) and at least one purified cellsurface receptor binding compound (e.g., IGF-1, EGF, NGF, and substanceP), for use as a supplement for organ storage solutions.

In some embodiments, the media described herein further comprise amicrotubule stabilizing agent selected from the group consisting oftaxol, discodermolide, epothilone A and B, vinblastine, andvinchristine.

In still further embodiments, the present invention provides methods andcompositions for stabilizing microtubules in cells, tissues, or organs,either in vitro, in vivo, or ex vivo. In preferred embodiments, thecompositions comprise a defensin (e.g., BNP-1). In other preferredembodiments, the compositions comprise a cell surface receptor bindingcompound, impermeant anion, energy source, or macromolecular oncoticagent as described in more detail above. In other particularly preferredembodiments, the present invention provides a composition comprising adefensin (e.g., BNP-1) for use in stabilizing microtubules and/or actinfilaments. In still other embodiments, the present invention providesmethods and processes comprising providing a cell, tissue or organ, anda composition comprising a purified defensin, and treating the cell,tissue, or organ under conditions such that the cytoskeleton of the celltissue, or organ is stabilized. In particularly preferred embodiments,microtubules and and/or actin filaments are stabilized. In still otherparticularly preferred embodiments, the defensin id BNP-1 (SEQ ID NO:37).

In still further embodiments, the present invention provides acomposition substantially as described in any of the examples herein.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for 3 days in UW solutionalone (solid line) or in UW solution supplemented with BNP-1 (dashedline).

FIG. 2 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for four days in UW solutionalone (solid circles), in UW solution supplemented with BNP-1 (solidsquares), or in UW solution supplemented with BNP-1 and growth factors(x's).

FIG. 3 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for four days in UW solutionalone (solid triangles) or six days in UW solution supplemented withtrophic factors (unfilled triangles).

FIG. 4 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for three days in UW solutionalone (solid tangles) or six days in UW solution supplemented withtrophic factors (squares).

FIG. 5 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for three days in UW solutionalone (squares) or five days in UW solution supplemented with trophicfactors (circles).

FIG. 6 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for three days in UW solutionalone (squares) or four days in UW solution supplemented with trophicfactors (diamonds).

FIG. 7 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for four days in UW solutionalone (solid triangles) or four days in UW solution supplemented withtrophic factors (diamonds).

FIG. 8 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for five days in UW solutionwith trophic factors and with starch (circles) or five days in UWsolution supplemented with trophic factors and without starch (squares).

FIG. 9 is a graph showing serum creatinine levels (Y-axis) over time(X-axis) in dogs receiving kidneys stored for three days in UW solutionsupplemented with BNP-1 (L-form isomer)(circles) or three days in UWsolution supplemented with BNP-1 (D-form isomer) (squares).

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “antimicrobial polypeptide” refers topolypeptides that inhibit the growth of microbes (e.g., bacteria).Examples of antimicrobial polypeptides include, but are not limited to,the polypeptides described in Table 1 below (e.g., defensins).Antimicrobial polypeptides include peptides synthesized from bothL-amino and D-amino acids.

As used herein, the term “pore forming agent” refers to any agent (e.g.,peptide or other organic compound) that forms pores in a biologicalmembrane. When the pore forming agent is a peptide, the peptide can besynthesized from both L-amino and D-amino acids.

As used herein, the term “cell surface receptor binding compound” refersto any compound that directly or indirectly (e.g., binding through anintermediate agent) binds to a cell surface receptor (e.g., an agonist).Cell surface receptor binding compounds can be proteins (e.g., IGF-1[insulin-like growth factor 1], IGF-2 [insulin-like growth factor 2],NGF-β[nerve growth factor-β], EGF [epidermal growth factor], CSGF[colony-stimulating growth factor], FGF [fibroblast growth factor], PDGF[platelet-derived growth factor], VEGF [vascular endothelial growthfactor], TGF-β [transforming growth factor β], and bone morphogeneticproteins), either purified from natural sources or geneticallyengineered, as well as fragments, mimetics, derivatives or modificationsthereof, and other organic compounds that bind to cell surface receptors(e.g., prostaglandins). Further examples of cell surface receptorbinding compounds are provided in U.S. Pat. Nos. 5,183,805; 5,218,093;5,130,298; 5,639,664; 5,457,034; 5,210,185; 5,470828; 5,650,496;5,998,376; and 5,410,019; all of which are incorporated herein byreference.

As used herein, the term “cellular material” refers to any material orcomposition comprising cells (e.g., cultured cells, gametes (i.e., spermand eggs), embryos, tissues, organs, and organisms).

As used herein, the term “internal organ” refers to an organ located inthe interior of the body (e.g., in the thoracic or abdominal cavity).Examples of internal organs include, but are not limited to kidneys,hearts, lungs, small intestines, large intestines, livers, andpancreases. Internal organs can be provided from a human donor (eithercadaveric or living) or be from an animal (e.g., for xenotransplants ortransplant studies in an animal model such as dogs).

As used herein, the term “delayed graft function” refers to the delay inthe return to normal serum creatinine following kidney transplant.

As used herein, the term “impermeant anion” refers to compounds thatcounteract swelling in organs that have been exposed to hypothermictemperatures. Examples of impermeant anions include, but are not limitedto, gluconate and lactobionic acid.

As used herein, the term “macromolecular oncotic agent” refers tocompounds used to maintain oncotic pressure equivalent to that of bloodplasma. Examples of macromolecular oncotic agents include, but are notlimited to, hydroxyethyl starch, dextran, trehalose, raffinose,mannitol, sucrose and glucose.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule expressed from a recombinant DNAmolecule. In contrast, the term “native protein” or “native polypeptide”is used herein to indicate a protein isolated from a naturally occurring(i.e., a nonrecombinant) source. Molecular biological techniques may beused to produce a recombinant form of a protein or polypeptide withsimilar or identical properties as compared to the native form of theprotein.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

As used herein in reference to an amino acid sequence or a protein, theterm “portion” (as in “a portion of an amino acid sequence”) refers tofragments of that protein. The fragments may range in size from fouramino acid residues to the entire amino acid sequence minus one aminoacid (e.g., 5, 6, 7, 8, . . . x−1).

As used herein, the term “variant,” when used in reference to a protein,refers to proteins encoded by partially homologous nucleic acids so thatthe amino acid sequence of the proteins varies. As used herein, the term“variant” encompasses proteins encoded by homologous genes having bothconservative and nonconservative amino acid substitutions that do notresult in a change in protein function, as well as proteins encoded byhomologous genes having amino acid substitutions that cause decreasedprotein function or increased protein function.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining the protein of interest (e.g., defensins and fragmentsthereof) joined to a heterologous protein fragment (e.g., the fusionpartner which consists of a non-defensin protein). The fusion partnermay enhance the solubility of a defensin as expressed in a host cell,may provide an affinity tag to allow purification of the recombinantfusion protein from the host cell or culture supernatant, or both. Ifdesired, the fusion protein may be removed from the protein of interest(e.g., defensin or fragments thereof) by a variety of enzymatic orchemical means know to the art.

As used herein, the term “purified” refers to molecules, either nucleicor amino acid sequences, that are removed from their naturalenvironment, isolated or separated. The percent of a purified componentis thereby increased in the sample. For example, an “isolated defensin”is therefore a purified defensin. “Substantially purified” molecules areat least 60% free, preferably at least 75% free, and more preferably atleast 90% free from other components with which they are naturallyassociated.

The term “gene” as used herein, refers to a DNA sequence that comprisescontrol and coding sequences necessary for the production of apolypeptide or protein precursor. The polypeptide can be encoded by afull length coding sequence or by any portion of the coding sequence, aslong as the desired protein activity is retained.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid. This situation is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologous to atarget under conditions of low stringency. This is not to say thatconditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be tested by the use of a secondtarget which lacks even a partial degree of complementarity (e.g., lessthan about 30% identity). In this case, in the absence of non-specificbinding, the probe will not hybridize to the second non-complementarytarget.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or a genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described herein.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acid strands. Hybridization and thestrength of hybridization (i.e., the strength of the association betweennucleic acid strands) is impacted by many factors well known in the artincluding the degree of complementarity between the nucleic acids,stringency of the conditions involved affected by such conditions as theconcentration of salts, the T_(m) (melting temperature) of the formedhybrid, the presence of other components (e.g., the presence or absenceof polyethylene glycol), the molarity of the hybridizing strands and theG:C content of the nucleic acid strands.

As used herein, the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “medium” or “low”stringency are often required when it is desired that nucleic acidswhich are not completely complementary to one another be hybridized orannealed together. The art knows well that numerous equivalentconditions can be employed to comprise medium or low stringencyconditions. The choice of hybridization conditions is generally evidentto one skilled in the art and is usually guided by the purpose of thehybridization, the type of hybridization (DNA-DNA or DNA-RNA), and thelevel of desired relatedness between the sequences (e.g., Sambrook etal., 1989, Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington D.C., 1985, for a general discussion of the state of theart).

The stability of nucleic acid duplexes is known to decrease with anincreased number of mismatched bases, and further to be decreased to agreater or lesser degree depending on the relative positions ofmismatches in the hybrid duplexes. Thus, the stringency of hybridizationcan be used to maximize or minimize stability of such duplexes.Hybridization stringency can be altered by: adjusting the temperature ofhybridization; adjusting the percentage of helix destabilizing agents,such as formamide, in the hybridization mix; and adjusting thetemperature and/or salt concentration of the wash solutions. For filterhybridizations, the final stringency of hybridizations often isdetermined by the salt concentration and/or temperature used for thepost-hybridization washes.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1× SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0× SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5× SSPE (43.8 μlNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5× SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature”. The melting temperature is the temperature at which 50% ofa population of double-stranded nucleic acid molecules becomesdissociated into single strands. The equation for calculating the Tm ofnucleic acids is well-known in the art. The T_(m) of a hybrid nucleicacid is often estimated using a formula adopted from hybridizationassays in 1 M salt, and commonly used for calculating T_(m) for PCRprimers: [(number of A+T)×2° C.+(number of G+C)×4° C.]. (C. R. Newton etal., PCR, 2nd Ed., Springer-Verlag (New York, 1997), p. 24). Thisformula was found to be inaccurate for primers longer than 20nucleotides. (Id.) Another simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl. (e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).Other more sophisticated computations exist in the art which takestructural as well as sequence characteristics into account for thecalculation of T_(m). A calculated T_(m) is merely an estimate; theoptimum temperature is commonly determined empirically.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another andcapable of replication in a cell. Vectors may include plasmids,bacteriophages, viruses, cosmids, and the like.

The terms “recombinant vector” and “expression vector” as used hereinrefer to DNA or RNA sequences containing a desired coding sequence andappropriate DNA or RNA sequences necessary for the expression of theoperably linked coding sequence in a particular host organism.Prokaryotic expression vectors include a promoter, a ribosome bindingsite, an origin of replication for autonomous replication in host cellsand possibly other sequences, e.g., an optional operator sequence. Apromoter is defined as a DNA sequence that directs RNA polymerase tobind to DNA and to initiate RNA synthesis. Eukaryotic expression vectorsinclude a promoter, polyadenlyation signal and optionally an enhancersequence.

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. Typically, the coding region is bounded on the 5′side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by a stop codon (e.g., TAA, TAG, TGA). Insome cases the coding region is also known to initiate by a nucleotidetriplet “TTG”.

The terms “buffer” or “buffering agents” refer to materials which whenadded to a solution, cause the solution to resist changes in pH.

The term “monovalent salt” refers to any salt in which the metal (e.g.,Na, K, or Li) has a net 1+ charge in solution (i.e., one more protonthan electron).

The term “divalent salt” refers to any salt in which a metal (e.g., Mg,Ca, or Sr) has a net 2+ charge in solution.

The term “solution” refers to an aqueous mixture.

The term “buffering solution” refers to a solution containing abuffering reagent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to media comprising antimicrobialpolypeptides and/or cell surface receptor binding compounds and theiruse for the storage and preservation of organs prior to transplant, andindeed, the preservation and storage of cellular materials in general.The media provided herein are superior to previously described media fororgan preservation. Animals receiving kidneys stored in the media of thepresent invention for either three or four days had serum creatininelevels of less than half of those observed in control animals receivingkidneys stored in UW solution alone. Therefore, it is contemplated thatthe use of the media of the present invention to preserve organs priorto transplant results both in improved function of the organ aftertransplant and an increase in the length of time for which the organscan be stored (i.e., increased storage capability).

Lowered serum creatinine levels are indicative of healthier kidneys anda more preferable prognosis for the transplant patient. It iscontemplated that transplant of healthier organs leads to a decrease inchronic rejection. Chronic rejection is a host versus graft rejectionthat occurs over a period of months to years, and is characterized byarterial and arteriolar thickening, atrophy, and fibrosis. Chronicrejection is the most common type of rejection for most solid organallografts. In fact, approximately ten percent of kidney transplantsfail each year due to chronic rejection. A 1999 survey indicates that amajority of transplant physicians and surgeons believe that chronicrejection is the area of transplant medicine that needs the mostimprovement (www.kidney.org/general/news/survey.cfm).

Additionally, use of the media of the present invention for cold storageor machine perfusion is expected to greatly reduce costs associated withdelayed graft function in kidneys. Most kidney transplant centerscurrently experience delayed graft function rates of between 20% and30%. When kidneys from non-beating heart donors are utilized, the rateof delayed graft function increases to approximately 75%-90%. Delayedgraft function has been estimated to add up to $20,000.00 to the cost ofa kidney transplant due to dialysis, complications, and longer hospitalstay. Furthermore, the incidence of delayed graft function is correlatedwith chronic rejection (i.e., 53% of kidneys in patients that needdialysis survive 5 years vs. 80% in optimal kidneys). The experimentaldata provided below in the Examples demonstrates that use of the mediacompositions of the present invention greatly reduces the time requiredto return to normal serum creatinine levels and thus reduces theincidence of delayed graft function.

Furthermore, it is expected that the media of the present invention willalso be useful for the storage and/or resuscitation of kidneys fromnon-beating heart donors so that they can routinely be used fortransplant. As described above, the delayed graft function ratesassociated with kidneys from non-beating heart donors exceeds 75%. Themajor source of delayed graft function of these kidneys is believed tobe warm ischemic injury. Most cold storage methods have been completelyunsuccessful in reducing preservation injury and delayed graft function.As a result, kidneys from non-beating heart donors that are subject towarm ischemic injury represent the largest untapped source of donorkidneys. It is contemplated that the use of the media of the presentinvention will facilitate routine use of kidneys from non-beating heartdonors, thus greatly expanding the pool of kidneys available torecipients. In particular, the use of the media of the present inventionto store kidneys from non-beating heart donors will result in a decreasein the delayed graft function rates normally observed when those kidneysare utilized for transplant.

Accordingly, improved compositions and methods for organ transplant aredescribed in detail below.

I. Transplant Media

The present invention contemplates the addition of antimicrobialpolypeptides (e.g., defensins) and/or cell surface receptor bindingcompounds to media used for organ transplantation and other proceduressuch as cardioplegia. In Section A, antimicrobial peptides useful in themedia of the present invention are described. In Section B, cell surfacereceptor binding compounds useful in the present invention aredescribed. In Section C, other components of organ transplantation mediaare described and representative formulas for organ preservation mediaare provided.

A. Antimicrobial Peptides

In some embodiments of the present invention, compositions forpreserving organs prior to transplantation are provided. In someembodiments of the present invention, media for preserving organscomprise one or more antimicrobial polypeptides (e.g., AntimicrobialPeptide Protocols, ed. W. M. Shafer, Humana Press, Totowa, N.J. [1997])or pore forming agents. In some embodiments, the antimicrobial peptideor pore forming agent is a compound or peptide selected from thefollowing: magainin (e.g., magainin I, magainin II, xenopsin, xenopsinprecursor fragment, caerulein precursor fragment), magainin I and IIanalogs (PGLa, magainin A, magainin G, pexiganin, Z-12, pexigaininacetate, D35, MSI-78A, MG0 [K10E, K11E, F12W-magainin 2], MG2+[K10E,F12W-magainin-2], MG4+[F12W-magainin 2], MG6+[f12W, E19Q-magainin 2amide], MSI-238, reversed magainin II analogs [e.g., 53D, 87-ISM, andA87-ISM], Ala-magainin II amide, magainin II amide), cecropin P1,cecropin A, cecropin B, indolicidin, nisin, ranalexin, lactoferricin B,poly-L-lysine, cecropin A (1-8)-magainin II (1-12), cecropin A(1-8)-melittin (1-12), CA(1-13)-MA(1-13), CA(1-13)-ME(1-13), gramicidin,gramicidin A, gramicidin D, gramicidin S, alamethicin, protegrin,histatin, dermaseptin, lentivirus amphipathic peptide or analog, parasinI, lycotoxin I or II, globomycin, gramicidin S, surfactin, ralinomycin,valinomycin, polymyxin B, PM2 [(+/−) 1-(4-aminobutyl)-6-benzylindane],PM2c [(+/−) -6-benzyl-1-(3-carboxypropyl)indane], PM3[(+/−)1-benzyl-6-(4-aminobutyl)indane], tachyplesin, buforin I or II,misgurin, melittin, PR-39, PR-26, 9-phenylnonylamine, (KLAKiKLA)n (SEOID NO: 97), (KLAKLAK)n (SEO ID NO: 98), where n=1, 2, or 3, (KALKALK)3(SEO ID NO: 99), KLGKKLG)n (SEO ID NO: 100), and KAAKKAA)n (SEO ID NO:101), wherein N+1, 2, or 3, paradaxin, Bac 5, Bac 7, ceratoxin, mdelin 1and 5, bombin-like peptides, PGQ, cathelicidin, HD-5, Oabac5alpha,ChBac5, SMAP-29, Bac7.5, lactoferrin, granulysin, thionin, hevein andknottin-like peptides, MPG 1, 1bAMP, snakin, lipid transfer proteins,and plant defensins. Exemplary sequences for the above compounds areprovided in Table 1. In some embodiments, the antimicrobial peptides aresynthesized from L-amino acids, while in other embodiments, the peptidesare synthesized from or comprise D-amino acids.

The compounds listed above can be isolated and purified from naturalsources as appropriate. The compounds may also be produced recombinantlyor synthetically as described below. In some embodiments, theantimicrobial peptide is included in the media at a concentrationsufficient to lower serum creatinine levels in kidney transplantrecipients as compared to recipients of kidneys stored withoutantimicrobial peptides. In other embodiments, the antimicrobialpolypeptide is included in the media at a concentration sufficient tocause a decrease in delayed graft function rates of kidneys stored inthe media as compared to unsupplemented media. Preferably, the time forreturn to baseline serum creatinine levels is improved by at least 25%,and most preferably by at least 50%, as compared to controlunsupplemented media. In preferred embodiments, the media of the presentinvention comprise one or more antimicrobial polypeptides at aconcentration of about 0.01 to 1000 mg/L. In particularly preferredembodiments, the media comprises a solution comprising one or moreantimicrobial polypeptides at a concentration of about 0.1 to 5 mg/L.

The present invention is not limited to a particular mechanism ofaction. Indeed, an understanding of the mechanism of action is notnecessary to practice the present invention. Nevertheless, the datasummarized in Example 10 demonstrates that the addition of anantimicrobial polypeptide to standard organ storage solutions (e.g., UWsolution) results in both the stabilization of cytoskeletal structureand an increased ability of the cytoskeleton to reassemble uponreperfusion. It is particularly notable that the antimicrobialpolypeptide stabilized both actin filaments and microtubules.

In some embodiments of the present invention, the antimicrobialpolypeptide is a defensin. In preferred embodiments, the compositions ofthe present invention comprise one or more defensins. In furtherpreferred embodiments, the composition comprises a solution comprisingpurified defensins at a concentration of about 0.01 to 1000 mg/L. Inparticularly preferred embodiments, the media comprises a solutioncomprising defensins at a concentration of about 0.1 to 5 mg/L. In stillfurther preferred embodiments, the antimicrobial polypeptide is BNP1(also known as bactanecin and bovine dodecapeptide). In certainembodiments, the defensin comprises the following consensus sequence:(SEQ ID NO:96 —X₁CN₁CRN₂CN₃ERN₄CN₅GN₆CCX₂, wherein N and X representconservatively or nonconservatively substituted amino acids and N₁=1,N₂=3 or 4, N₃=3 or 4, N₄=1, 2, or 3, N₆=5-9, X₁ and X₂ may be present,absent, or equal from 1-2.

The present invention is not limited to any particular defensin. Indeed,media comprising a variety of defensins are contemplated. Representativedefensins are provided in Tables 1 and 2 below. In general, defensinsare a family of highly cross-linked, structurally homologousantimicrobial peptides found in the azurophil granules ofpolymorphonuclear leukocytes (PMN's) with homologous peptides beingpresent in macrophages (e.g., Selsted et al., Infect. Immun. 45:150-154[1984]). Originally described as “Lysosomal Cationic Peptides” in rabbitand guinea pig PMN (Zeya et al., Science 154:1049-1051 [1966]; Zeya etal., J. Exp. Med. 127:927-941 [1968]; Zeya et al., Lab. Invest.24:229-236 [1971]; Selsted et al., [1984], supra.), this mixture wasfound to account for most of the microbicidal activity of the cruderabbit PMN extract against various microorganisms (Zeya et al., [1966],supra; Lehrer et al., J. Infect. Dis. 136:96-99 [1977]; Lehrer et al.,Infect. Immun. 11:1226-1234 [1975]). Six rabbit neutrophil defensinshave been individually purified and are designated NP-1, NP-2, NP-3A,NP-3B, NP-4, and NP-5. Their amino acid sequences were determined, andtheir broad spectra of activity were demonstrated against a number ofbacteria (Selsted et al., Infect. Immun. 45:150-154 [1984]), viruses(Lehrer et al., J. Virol. 54:467 [1985]), and fungi (Selsted et al.,Infect. Immun. 49:202-206 [1985]; Segal et al., 151:890-894 [1985]).Defensins have also been shown to possess mitogenic activity (e.g.,Murphy et al., J. Cell. Physiol. 155:408-13 [1993]).

Four peptides of the defensin family have been isolated from human PMN'sand are designated HNP-1, HNP-2, HNP-3, and HNP-4 (Ganz et al., J. Clin.Invest. 76:1427-1435 [1985]; Wilde et al., J. Biol. Chem.264:11200-11203 [1989]). The amino acid sequences of HNP-1, HNP-2, andHNP-3 differ from each other only in their amino terminal residues,while each of the human defensins are identical to the six rabbitpeptides in 10 or 11 of their 29 to 30 residues. These are the same 10or 11 residues that are shared by all six rabbit peptides. Humandefensin peptides have been shown to share with the rabbit defensins abroad spectrum of antimicrobial activity against bacteria, fungi, andenveloped viruses (Ganz et al., [1985], supra).

Three defensins designated RatNP-1, RatNP-2, and RatNP-4, have beenisolated from rat (Eisenhauer et al., Infection and Immunity57:2021-2027 [1989]). A guinea pig defensin (GPNP) has also beenisolated, purified, sequenced and its broad spectrum antimicrobialproperties verified (Selsted et al., Infect. Immun. 55:2281-2286[1987]). Eight of its 31 residues were among those invariant in sixrabbit and three human defensin peptides. The sequence of GPNP alsoincluded three nonconservative substitutions in positions otherwiseinvariant in the human and rabbit peptides. Of the defensins tested in aquantitative assay HNP-1, RatNP-1, and rabbit NP-1 possess the mostpotent antimicrobial properties, while NP-5 possesses the least amountof antimicrobial activity when tested against a panel of organisms instationary growth phase (Selsted et al., Infect. Immun. 45:150-154[1984]; Ganz et al., J. Clin. Invest. 76:1427-1435 [1985]). Defensinpeptides are further described in U.S. Pat. Nos. 4,543,252; 4,659,692;and 4,705,777 (each of which is incorporated herein by reference).

Accordingly, in some embodiments, the media comprises one or moredefensins selected from the group consisting of SEQ ID NOs: 37-95. Inparticularly preferred embodiments, the media comprises bovine defensinpeptide (BNP-1; SEQ ID NO: 37, Romeo et al., J. Biol. Chem.263(15):9573-9575 [1988]). In some embodiments, the defensin is includedin the media at a concentration sufficient to lower serum creatininelevels in kidney transplant recipients as compared to recipients ofkidneys stored without defensin peptides.

Defensin peptides suitable for use in the methods and compositions ofthe present invention include natural defensin peptides isolated fromknown cellular sources, synthetic peptides produced by solid phase orrecombinant DNA techniques, and defensin analogs which may be smallerpeptides or other molecules having similar binding and biologicalactivity as the natural defensin peptides (e.g., peptide mimetics).Methods for the purification of defensin peptides are described in U.S.Pat. Nos. 4,543,252; 4,659,692; and 4,705,777, the disclosures of whichare incorporated herein by reference.

In preferred embodiments, suitable synthetic peptides will usuallycomprise all or part of the amino acid sequence of a known peptide, morepreferably incorporating at least some of the conserved regionsidentified in Table 2. In particularly preferred embodiments, thesynthetic peptides incorporate at least one of the conserved regions,more usually incorporating two of the conserved regions, preferablyconserving at least three of the conserved regions, and more preferablyconserving four or more of the conserved regions. In preferredembodiments, the synthetic peptides comprise fifty amino acids or fewer,although there may be advantages in increasing the size of the peptideabove that of the natural peptides in certain instances. In certainembodiments, the peptides have a length in the range from about 10 to 50amino acids, preferably being in the range from about 10 to 40 aminoacids, and most preferably being in the range from about 30 to 35 aminoacids which corresponds generally to the length of the natural defensinpeptides.

In some cases, it may be desirable to incorporate one or morenon-natural amino acids in the synthetic defensin peptides of thepresent invention. In preferred embodiments, non-natural amino acidscomprise at least an N-terminus and a C-terminus and have side chainsthat are either identical to or chemically modified or substituted froma natural amino acid counterpart. An example of a non-natural amino acidis an optical isomer of a naturally-occurring L-amino acid, such as apeptide containing all D-amino acids. Examples of the synthesis ofpeptides containing all D-amino acids include Merrifield et al., CibaFound Symp. 186:5-26 (1994); Wade et al., Proc. Natl. Acad. Sci. USA87(12):4761-5 (1990); and U.S. Pat. No. 5,792,831, which is hereinincorporated by reference. Examples of chemical modifications orsubstitutions include hydroxylation or fluorination of C—H bonds withinnatural amino acids. Such techniques are used in the manufacture of druganalogs of biological compounds and are known to one of ordinary skillin the art.

Synthetic peptides having biological and binding activity the same orsimilar to that of natural defensin peptides may be produced by eitherof two exemplary approaches. First, the polypeptides may be produced bythe well-known Merrifield solid-phase chemical synthesis method whereinamino acids are sequentially added to a growing chain (Merrifield (1963)J. Am. Chem. Soc. 85:2149-2156 [1963]). Automatic peptide synthesisequipment is available from several commercial suppliers, including PEBiosystems, Inc., Foster City, Calif.; Beckman Instruments, Inc.,Waldwick, N.J.; and Biosearch, Inc., San Raphael, Calif. Using suchautomatic synthesizers according to manufacturer's instructions,peptides may be produced in gram quantities for use in the presentinvention.

Second, the synthetic defensin peptides of the present invention may besynthesized by recombinant techniques involving the expression incultured cells of recombinant DNA molecules encoding a gene for adesired portion of a natural or analog defensin molecule. The geneencoding the defensin peptide may itself be natural or synthetic.Conveniently, polynucleotides may be synthesized by well knowntechniques based on the desired amino acid sequence. For example, shortsingle-stranded DNA fragments may be prepared by the phosphoramiditemethod (Beaucage et al., Tetra. Lett. 22:1859-1862 [1981]). Adouble-stranded fragment may then be obtained either by synthesizing thecomplementary strand and annealing the strands together underappropriate conditions or by adding the complementary strand using DNApolymerase under appropriate conditions or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence. Thenatural or synthetic DNA fragments coding for the desired defensinpeptide may then be incorporated in a suitable DNA construct capable ofintroduction to and expression in an in vitro cell culture. The DNAfragments can be portions or variants of wild-type nucleic acidsencoding defensins. Suitable variants include those both withconservative and nonconservative amino acid substitutions.

The methods and compositions of the present invention may also employsynthetic non-peptide compositions that have biological activityfunctionally comparable to that of the known defensin peptides. Byfunctionally comparable, it is meant that the shape, size, flexibility,and electronic configuration of the non-peptide molecule is such thatthe biological activity of the molecule is similar to the defensinpeptides. In particular, the non-peptide molecules should displaycomparable mitogenic activity and/or antimicrobial activity or poreforming ability, preferably possessing both activities. Such non-peptidemolecules will typically be small molecules having a molecular weight inthe range from about 100 to 1000 daltons. The use of such smallmolecules is frequently advantageous in the preparation ofpharmacological compositions. Candidate mimetics can be screened inlarge numbers to identify those having the desired activity.

The identification of such nonpeptide analog molecules can be performedusing techniques known in the art of drug design. Such techniquesinclude, but are not limited to, self-consistent field (SCF) analysis,configuration interaction (CI) analysis, and normal mode dynamicscomputer analysis, all of which are well described in the scientificliterature (Rein et al., Computer-Assisted Modeling of Receptor-LigandInteractions, Alan Liss, N.Y., [1989]). Preparation of the identifiedcompounds will depend on the desired characteristics of the compoundsand will involve standard chemical synthetic techniques (Cary et al.,Advanced Organic Chemistry, part B, Plenum Press, New York [1983]).

TABLE 1 Antimicrobial Peptides SEQ ID NO: Name Organism Sequence  1lingual antimicrobial Bos taurusmrlhhlllallflvlsagsgftqgvrnsqscrrnkgicvp peptide precursorircpgsmrqigtclgaqvkccrrk (Magainin)  2 antimicrobial peptide Xenopuslaevis gvlsnvigylkklgtgalnavlkq PGQ  3 Xenopsin Xenopus laevismykgiflcvllavicanslatpssdadedndeveryvrgwaskigqtlgkiakvglkeliqpkreamlrsaeaqgkrpwil  4 magainin precursor Xenopuslaevis mfkglficsliavicanalpqpeasadedmderevrgigkflhsagkfgkafvgeimkskrdaeavgpeafadedlderevrgigkflhsakkfgkafvgeimnskrdaeavgpeafadedlderevrgigkflhsakkfgkafvgeimnskrdaeavgpeafadedlderevrgigkflhsakkfgkafvgeimnskrdaeavgpeafadedfderevrgigkflhsakkfgkafvgeimnskrdaeavgpeafadedlderevrgigkflhsakkfgk afvgeimnskrdaeavddrrwve  5tachyplesin I Tachypleus kwcfrvcyrgicyrrcr gigas  6 tachyplesin IITachypleus rwcfrvcyrgicyrkcr gigas  7 buforin I Bufo bufomsgrgkqggkvrakaktrssraglqfpvgrvhrllrkgny gagarizansaqrvgagapvylaavleyltaeilelagnaardnkktriiprhlqlavrndeelnkllggvtiaqggvlpniqavllpkt esskpaksk  8 buforin II Bufobufo trssraglqfpvgrvhrllrk gagarizans  9 cecropin A Bombyx morimnfvrilsfvfalvlalgavsaapeprwklfkkiekvgrn vrdglikagpaiavigqakslgk 10cecropin B Bombyx mori mnfakilsfvfalvlalsmtsaapeprwkifkkiekmgrnirdgivkagpaievlgsakaigk 11 cecropin C Drosophilamnfykifvfvalilaisigqseagwlkklgkrierigqht melanogasterrdatiqglgiaqqaanvaatarg 12 cecropin P1 Sus scrofaswlsktakklensakkrisegiaiaiqggpr 13 indolicidin Bos taurus ilpwkwpwwpwrr14 nisin Lactococcus itsislctpgcktgalmgcnmktatchcsihvsk lactis 15ranalexin Rana flgglikivpamicavtkkc catesbeiana 16 lactoferricin B Bostaurus fkcrrwqwrmkklgapsitcvrraf 17 protegrin-1 Sus scrofarggrlcycrrrfcvcvgrx 18 protegrin-2 Sus scrofa ggrlcycrrrfcicvg 19histatin precursor Homo sapiens mkffvfalilalmlsmtgadshakrhhgykrkfhekhhshrgyrsnylydn 20 histatin 1 Macaca dsheerhhgrhghhkygrkfhekhhshrgyrsnylydnfascicularis 21 dermaseptin Phyllomedusaalwktmlkklgtmalhagkaalgaaadtisqtq sauvagei 22 dermaseptin 2 Phyllomedusaalwftmlkklgtmalhagkaalgaaantisqgtq sauvagei 23 dermaseptin 3Phyllomedusa alwknmlkgigklagkaalgavkklvgaes sauvagei 24 misgurinMisgurnus rqrveelskfskkgaaarrrk anguillicaudatus 25 melittin Apismellifera gigavlkvlttglpaliswisrkkrqq 26 pardaxin-1 Pardachirusgffalipkiissplfktllsavgsalsssgeqe pavoninus 27 pardaxin-2 Pardachirusgffalipkiisspifktllsavgsalsssggqe pavoninus 28 bactenecin 5 precursorBos taurus metqraslslgrcslwllllglvlpsasaqalsyreavlravdqfnersseanlyrlleldptpnddldpgtrkpvsfrvketdcprtsqqpleqcdfkenglvkqcvgtvtldpsndqfdincnelqsvrfrppirrppirppfyppfrppirppifpp irppfrpplgpfpgrr 29 bactenecinprecursor Bos taurus metpraslslgrwslwllllglalpsasaqalsyreavlravdqlneqssepniyrlleldqppqddedpdspkrvsfrvketvcsrttqqppeqcdfkengllkrcegtvtldqvrgnfditcnnhqsiritkqpwappqaarlcrivvirvcr 30 ceratotoxin A Ceratitissigsalkkalpvakkigkialpiakaalp capitata 31 ceratotoxin B Ceratitissigsafkkalpvakkigkaalpiakaalp capitata 32 cathelicidin antimicrobialHomo sapiens mktqrnghslgrwslvllllglvmplaiiaqvlsykeavl peptideraidginqrssdanlyrlldldprptmdgdpdtpkpvsftvketvcprttqqspedcdfkkdglvkrcmgtvtlnqargsfdiscdkdnkrfallgdffrkskekigkefkrivqrikdf lrnlvprtes 33 myeloidcathelicidin 3 Equus caballus metqrntrclgrwsplllllglvippattqalsykeavlravdglnqrssdenlyrlleldplpkgdkdsdtpkpvsfmvketvcprimkqtpeqcdfkenglvkqcvgtvildpvkdyfdascdepqrvkrfhsvgsliqrhqqmirdkseatrhgiri itrpklllas 34 myeloidantimicrobial Bos taurus metqraslslgrwslwllllglalpsasaqalsyreavlrpeptide BMAP-28 avdqlneksseanlyrlleldpppkeddenpnipkpvsfrvketvcprtsqqspeqcdfkengllkecvgtvtldqvgsnfditcavpqsvgglrslgrkilrawkkygpiivpiirig 35 myeloid cathelicidin 1 Equuscaballus metqrntrclgrwsplllllglvippattqalsykeavlravdglnqrssdenlyrlleldplpkgdkdsdtpkpvsfmvketvcprimkqtpeqcdfkenglvkqcvgtvilgpvkdhfdvscgepqrvkrfgrlaksflrmrillprrkillas 36 SMAP 29 Ovis ariesmetqraslslgrcslwllllglalpsasaqvlsyreavlraadqlneksseanlyrlleldpppkqddensnipkpvsfrvketvcprtsqqpaeqcdfkengllkecvgtvtldqvrnnfditcaepqsvrglrrlgrkiahgvkkygptvlriiriag 37 BNP-1 Bos taurusrlcrivvirvcr 38 HNP-1 Homo sapiens acycripaciagerrygtciyqgrlwafcc 39HNP-2 Homo sapiens cycripaciagerrygtciyqgrlwafcc 40 HNP-3 Homo sapiensdcycripaciagerrygtciyqgrlwafcc 41 HNP-4 Homo sapiensvcscrlvfcrrtelrvgncliggvsftycctrv 42 NP-1 Oryctolagusvvcacrralclprerragfcrirgrihplccrr cuniculus 43 NP-2 Oryctolagusvvcacrralclplerragfcrirgrihplccrr cuniculus 44 NP-3A Oryctolagusgicacrrrfcpnserfsgycrvngaryvrccsrr cuniculus 45 NP-3B Oryctolagusgrcvcrkqllcsyrerrigdckirgvrfpfccpr cuniculus 46 NP-4 Oryctolagusvsctcrrfscgfgerasgsctvnggvrhtlccrr cuniculus 47 NP-5 Oryctolagusvfctcrgflcgsgerasgsctingvrhtlccrr cuniculus 48 RatNP-1 Rattusvtcycrrtrcgfrerlsgacgyrgriyrlccr norvegicus 49 Rat-NP-3 Rattuscscrysscrfgerllsgacrlngriyrlcc norvegicus 50 Rat-NP-4 Rattusactcrigacvsgerltgacglngriyrlccr norvegicus 51 GPNP Guinea pigrrcicttrtcrfpyrrlgtcifqnrvytfcc 52 beta defensin-3 Homo sapiensmrihyllfallflflvpvpghggiintlqkyycrvrggrc avlsclpkeeqigkcstrgrkccrrkk 53theta defensin-1 Macaca rcictrgfcrclcrrgvc mulatta 54 defensin CUA1Helianthus mkssmkmfaalllvvmcllanemggplvveartcesqshk annuusfkgtclsdtncanvchserfsggkcrgfrrrcfctthc 55 defensin SD2 Helianthusmkssmkmfaalllvvmcllanemggplvveartcesqshk annuusfkgtclsdtncanvchserfsggkcrgfrrrcfctthc 56 neutrophil defensin 2 Macacaacycripaclagerrygtcfymgrvwafcc mulatta 57 4 KDA defensin Androctonusgfgcpfnqgachrhcrsirrrggycaglfkqtctcyr australis hector 58 defensinMytilus gfgcpnnyqchrhcksipgrcggycggxhrlrctcyrc galloprovincialis 59defensin AMP1 Heuchera dgvklcdvpsgtwsghcgssskcsqqckdrehfayggachsanguinea yqfpsvkcfckrqc 60 defensin AMP1 Clitorianlcerasltwtgncgntghcdtqcrnwesakhgachkrgn ternatea wkcfcyfnc 61cysteine-rich cryptdin-1 Mus musculusmkklvllfalvllafqvqadsiqntdeetkteeqpgekdq homologavsvsfgdpqgsalqdaalgwgrrcpqcprcpscpscprc prcprckcnpk 62 beta-defensin-9Bos taurus qgvrnfvtcrinrgfcvpircpghrrqigtclgpqikccr 63 beta-defensin-7Bos taurus qgvrnfvtcrinrgfcvpircpghrrqigtclgprikccr 64 beta-defensin-6Bos taurus qgvrnhvtcriyggfcvpircpgrtrqigtcfgrpvkccrrw 65 beta-defensin-5Bos taurus qvvrnpqscrwnmgvcipiscpgnmrqigtcfgprvpccr 66 beta-defensin-4Bos taurus qrvrnpqscrwnmgvcipflcrvgmrqigtcfgprvpccrr 67 beta-defensin-3Bos taurus qgvrnhvtcrinrgfcvpircpgrtrqigtcfgprikccrsw 68beta-defensin-10 Bos taurus qgvrsylscwgnrgicllnrcpgrmrqigtclaprvkccr 69beta-defensin-13 Bos taurus sgisgplscgrnggvcipircpvpmrqigtcfgrpvkccrsw70 beta-defensin-1 Bos taurus dfaschtnggiclpnrcpghmiqigicfrprvkccrsw 71coleoptericin Zophobas slqggapnfpqpsqqnggwqvspdlgrddkgntrgqieiq atratusnkgkdhdfnagwgkvirgpnkakptwhvggtyrr 72 beta defensin-3 Homo sapiensmrihyllfallflflvpvpghggiintlqkyycrvrggrc avlsclpkeeqigkcstrgrkccrrkk 73defensin C Aedes aegypti atcdllsgfgvgdsacaahciargnrggycnskkvcvcrn 74defensin B Mytilus edulis gfgcpndypchrhcksipgryggycggxhrlrctc 75 sapecinC Sarcophaga atcdllsgigvqhsacalhcvfrgnrggyctgkgicvcrn peregrina 76macrophage antibiotic Oryctolagusmrtlallaaillvalqaqaehvsvsidevvdqqppqaedq peptide MCP-1 cuniculusdvaiyvkehessalealgvkagvvcacrralclprerrag fcrirgrihplccrr 77 cryptdin-2Mus musculus mkplvllsalvllsfqvqadpiqntdeetkteeqsgeedqavsvsfgdregaslqeeslrdlvcycrtrgckrrermngt crkghlmytlcc 78 cryptdin-5 Musmusculus mktfvllsalvllafqvqadpihktdeetnteeqpgeedqavsisfggqegsalheelskklicycrirgckrrervfgt crnlfltfvfccs 79 cryptdin 12Mus musculus lrdlvcycrargckgrermngtcrkghllymlccr 80 defensin Pyrrhocorisatcdilsfqsqwvtpnhagcalhcvikgykggqckitvchcrr apterus 81 defensin R-5Rattus vtcycrstrcgfrerlsgacgyrgriyrlccr norvegicus 82 defensin R-2Rattus vtcscrtsscrfgerlsgacrlngriyrlcc norvegicus 83 defensin NP-6Oryctolagus gicacrrrfclnfeqfsgycrvngaryvrccsrr cuniculus 84beta-defensin-2 Pan mrvlyllfsflfiflmplpgvfggisdpvtclksgaichp troglodytesvfcprrykqigtcglpgtkcckkp 85 beta-defensin-2 Homo sapiensmrvlyllfsflfiflmplpgvfggigdpvtclksgaichp vfcprrykqigtcglpgtkcckkp 86beta-defensin-1 Homo sapiens mrtsylllftlclllsemasggnfltglghrsdhyncvssggqclysacpiftkiqgtcyrgkakcck 87 beta-defensin-1 Capra hircusmrlhhlllvlfflvlsagsgftqgirsrrschrnkgvcal trcprnmrqigtcfgppvkccrkk 88beta defensin-2 Capra hircus mrlhhlllalfflvlsagsgftqgiinhrscyrnkgvcaparcprnmrqigtchgppvkccrkk 89 defensin-3 Macacamrtlvilaaillvalqaqaeplqartdeataaqeqiptdn mulattapevvvslawdeslapkdsvpglrknmacycripaclager rygtcfyrrrvwafcc 90 defensin-1Macaca mrtlvilaaillvalqaqaeplqartdeataaqeqiptdn mulattapevvvslawdeslapkdsvpglrknmacycripaclager rygtcfylgrvwafcc 91 neutrophildefensin 1 Mesocricetus vtcfcrrrgcasrerhigycrfgntiyrlccrr auratus 92neutrophil defensin 1 Mesocricetus cfckrpvcdsgetqigycrlgntfyrlccrqauratus 93 Gallinacin 1-alpha Gallus gallusgrksdcfrkngfcaflkcpyltlisgkcsrfhlcckriw 94 defensin Allomyrinavtcdllsfeakgfaanhslcaahclaigrrggscergvcicrr dichotoma 95 neutrophilcationic Cavia rrcicttrtcrfpyrrlgtcifqnrvytfcc peptide 1 porcellus

TABLE 2 Defensins SEQ ID NO Name Organism Sequence 38 HNP-1 HumanACYCRIPACIAGERRYGTCIYQGRLWAFCC 39 HNP-2 HumanCYCRIPACIAGERRYGTCIYQGRLWAFCC 40 HNP-3 HumanDCYCRIPACIAGERRYGTCIYQGRLWAFCC 41 HNP-4 HumanVCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRV 42 NP-1 RabbitVVCACRRALCLPRERRAGFCRIRGRIHPLCCRR 43 NP-2 RabbitVVCACRRALCLPLERRAGFCRIRGRIHPLCCRR 44 NP-3A RabbitGICACRRRFCPNSERFSGYCRVNGARYVRCCSRR 45 NP-3B RabbitGRCVCRKQLLCSYRERRIGDCKIRGVRFPFCCPR 46 NP-4 RabbitVSCTCRRFSCGFGERASGSCTVNGVRHTLCCRR 47 NP-5 RabbitVFCTCRGFLCGSGERASGSCTINGVRHTLCCRR 48 RatNP-1 RatVTCYCRRTRCGFRERLSGACGYRGRIYRLCCR 49 Rat-NP-3 RatCSCRYSSCRFGERLLSGACRLNGRIYRLCC 50 Rat-NP-4 RatACTCRIGACVSGERLTGACGLNGRIYRLCCR 51 GPNP Guinea pigRRCICTTRTCRFPYRRLGTCIFQNRVYTFCCB. Cell Surface Receptor Binding Compounds

In some embodiments of the present invention, media for preservingorgans comprise one or more cell surface receptor binding compounds.Cell surface receptor binding compounds useful in the present inventioninclude, but are not limited to, the following broad classes ofcytoactive compounds: Insulin, Insulin like Growth Factors such asIGF-I, IGF-II, and IGF-BP; Epidermal Growth Factors such as α-EGF andβ-EGF; EGF-like molecules such as Keratinocyte-derived growth factor(which is identical to KAF, KDGF, and amphiregulin) and vaccinia virusgrowth factor (VVGF); Fibroblast Growth Factors such as FGF-1 (Basic FGFProtein), FGF-2 (Acidic FGF Protein), FGF-3 (Int-2), FGF-4 (Hst-1),FGF-5, FGF-6, and FGF-7 (identical to KGF); FGF-Related Growth Factorssuch as Endothelial Cell Growth Factors (e.g., ECGF-α and ECGF-β); FGF—and ECGF-Related Growth Factors such as Endothelial cell stimulatingangiogenesis factor and Tumor angiogenesis factor, Retina-Derived GrowthFactor (RDGF), Vascular endothelium growth factor (VEGF), Brain-DerivedGrowth Factor (BDGF A- and -B), Astroglial Growth Factors (AGF 1 and 2),Omentum-derived factor (ODF), Fibroblast-Stimulating factor (FSF), andEmbryonal Carcinoma-Derived Growth Factor; Neurotrophic Growth Factorssuch as α-NGF, β-NGF, γ-NGF, Brain-Derived Neurotrophic Factor (BDNF),Neurotrophin-3, Neurotrophin-4, and Ciliary Nuerotrophic Factor (CNTF);Glial Growth Factors such as GGF-I, GGF-II, GGF-III, Glia MaturationFactor (GMF), and Glial-Derived Nuerotrophic Factor (GDNF);Organ-Specific Growth Factors such as Liver Growth Factors (e.g.,Hepatopoietin A, Hepatopoietin B, and Hepatocyte Growth Factors (HCGF orHGF), Prostate Growth Factors (e.g., Prostate-Derived Growth Factors[PGF] and Bone Marrow-Derived Prostate Growth Factor), Mammary GrowthFactors (e.g., Mammary-Derived Growth Factor 1 [MDGF-1] and MammaryTumor-Derived Factor [MTGF]), and Heart Growth Factors (e.g.,Nonmyocyte-Derived Growth Factor [NMDGF]); Cell-Specific Growth Factorssuch as Melanocyte Growth Factors (e.g., Melanocyte-Stimulating Hormone[α-, β-, and γ-MSH] and Melanoma Growth-Stimulating Activity [MGSA]),Angiogenic Factors (e.g., Angiogenin, Angiotropin, Platelet-DerivedECGF, VEGF, and Pleiotrophin), Transforming Growth Factors (e.g., TGF-α,TGF-β, and TGF-like Growth Factors such as TGF-β₂, TGF-β₃, TGF-e, GDF-1,CDGF and Tumor-Derived TGF-β-like Factors), ND-TGF, and Human epithelialtransforming factor [h-TGFe]); Regulatory Peptides with GrowthFactor-like Properties such as Bombesin and Bombesin-like peptides(e.g., Ranatensin, and Litorin], Angiotensin, Endothelin, AtrialNatriuretic Factor, Vasoactive Intestinal Peptide, and Bradykinin;Cytokines such as the interleukins IL-1 (e.g., Osteoclast-activatingfactor [OAF], Lymphocyte-activating factor [LAF], Hepatocyte-stimulatingfactor [HSF], Fibroblast-activating factor [FAF], B-cell-activatingfactor [BAF], Tumor inhibitory factor 2 [TIF-2], Keratinocyte-derivedT-cell growth factor [KD-TCGF]), IL-2 (T-cell growth factor [TCGF],T-cell mitogenic factor [TCMF]), IL-3 (e.g., Hematopoietin,Multipotential colony-stimulating factor [multi-CSF], Multilineagecolony-stimulating activity [multi-CSA], Mast cell growth factor [MCGF],Erythroid burst-promoting activity [BPA-E], IL-4 (e.g., B-cell growthfactor I [BCGF-I], B-cell stimulatory factor 1 [BSF-1]), IL-5 (e.g.,B-cell growth factor II [BCGF-II], Eosinophil colony-stimulating factor[Eo-CSF], Immunoglobulin A-enhancing factor [IgA-EF], T-cell replacingfactor [TCRF]), IL-6 (B-cell stimulatory factor 2 [BSF-2], B-cellhybridoma growth factor [BCHGF], Interferon β₂ [IFN-B], T-cellactivating factor [TAF], IL-7 (e.g., Lymphopoietin 1 [LP-1], Pre-B-cellgrowth factor [pre-BCGF]), IL-8 (Monocyte-derived neutrophil chemotacticfactor [MDNCF], Granulocyte chemotatic factor [GCF],Neutrophil-activating peptide 1 [NAP-1], Leukocyte adhesion inhibitor[LAI], T-lymphocyte chemotactic factor [TLCF]), IL-9 (e.g., T-cellgrowth factor III [TCGF-III], Factor P40, MegaKaryoblast growth factor(MKBGF), Mast cell growth enhancing activity [MEA or MCGEA]), IL-10(e.g., Cytokine synthesis inhibitory factor [CSIF]), IL-11 (e.g.,Stromal cell-derived cytokine [SCDC]), IL-12 (e.g., Natural killer cellstimulating factor [NKCSF or NKSF], Cytotoxic lymphocyte maturationfactor [CLMF]), TNF-α (Cachectin), TNF-β (Lymphotoxin), LIF(Differentiation-inducing factor [DIF], Differentiation-inducingactivity [DIA], D factor, Human interleukin for DA cells [HILDA],Hepatocyte stimulating factor III [HSF-III], Cholinergic neuronaldifferentiation factor [CNDF], CSF-1 (Macrophage colony-stimulatingfactor [M-CSF]), CSF-2 (Granulocyte-macrophage colony-stimulating factor[GM-CSF]), CSF-3 (Granulocyte colony-stimulating factor [G-CSF]), anderythropoietin; Platelet-derived growth factors (e.g., PDGF-A, PDGF-B,PDGF-AB, p28-sis, and p26-cis), and Bone Morphogenetic protein (BMP),neuropeptides (e.g., Substance P, calcitonin gene-regulated peptide, andneuropeptide Y), and neurotransmitters (e.g., norepinephrine andacetylcholine).

In some preferred embodiments, EGF, IGF-1, and/or NGF are included inthe media at a concentration of about 1 ng/ml to 100 ng/ml, mostpreferably about 10 ng/ml. In other preferred embodiments, substance Pis included at a concentration of about 0.1 μg/ml to 100 μg/ml, mostpreferably about 2.5 μg/ml. In some embodiments, NGF is deleted as itmay not be essential for suppressing delayed graft function. In someembodiments, the cell surface receptor binding compound is included inthe media at a concentration sufficient to lower serum creatinine levelsin kidney transplant recipients as compared to recipients of kidneysstored without cell surface receptor binding compounds. In otherembodiments, the cell surface receptor binding compound(s) are includedin the media at concentrations sufficient to cause a decrease in delayedgraft function rates of kidneys stored in the media as compared tounsupplemented media. Preferably, the time for return to baseline serumcreatinine levels is improved by at least 25%, and most preferably by atleast 50%, as compared to control unsupplemented media.

Suitable cell surface receptor binding compounds may be obtained fromcommercial sources, purified from natural sources, or be produced byrecombinant methods. Recombinant cell surface receptor binding compoundscan be produced from wild-type coding sequences or from variantsequences that encode functional cell surface receptor bindingcompounds. Suitable cell surface receptor binding compounds also includeanalogs which may be smaller peptides or other molecules having similarbinding and biological activity as the natural cell surface receptorbinding compounds. Methods for producing cell surface receptor bindingcompounds are described in U.S. Pat. Nos. 5,183,805; 5,218,093;5,130,298; 5,639,664; 5,457,034; 5,210,185; 5,470828; 5,650,496;5,998,376; and 5,410,019; all of which are incorporated herein byreference.

C. Other Transplant Media Components

In certain embodiments, a number of other components are utilized in themedia of the present invention to provide the proper balance ofelectrolytes, a physiological pH, proper oncotic pressure, etc.Therefore, it is contemplated that the media comprises one or morecomponents selected from one or more of the following general groups: 1)electrolytes; 2) oncotic agents; 3) buffers; 4) energy sources; 5)impermeant anions; 6) free radical scavengers; and/or 7) ATP sources.Examples of these components are provided below along with severalexemplary media formulations. Examples of media that can be supplementedwith defensins include VIASPAN (U.S. Pat. Nos. 4,798,824; 4,873,230; and5,696,152, each of which is incorporated herein by reference) andvarious HYPOTHERMOSOL formulations (U.S. Pat. Nos. 5,514,536 and6,045,990, each of which is incorporated herein by reference).

1) Electrolytes

In some embodiments of the present invention, the media compriseselectrolytes (e.g., sodium, potassium, calcium, magnesium, chloride,sulfate, bicarbonate, and phosphate) in concentrations approximatingthose found in blood plasma. For example, in some embodiments, potassiumand phosphate are provided as KH₂PO₄ in range from about 10 to 50 mM,preferably about 25 mM; magnesium is provided as magnesium gluconate ina range of from about 1 to 10 mM, preferably about 5 mM; sodium isprovided as sodium gluconate in a range of from about 50 mM to about 150mM, preferably about 105 mM; and calcium and chloride are provided asCaCl₂ in a range of from about 0.1 to 5.0 mM, preferably about 0.5 mM.

In other embodiments, the concentration of individual electrolytes maybe varied from physiological concentrations. For example, it is knownthat membrane pumps of cells are turned off during hypothermia. As aresult, potassium and sodium exchange passively across the cellmembrane. The media can be adjusted to compensate for the influx ofsodium and efflux of potassium by providing potassium in a range of fromabout 35 to 45 mM and sodium in a range of from about 80 to 120 mM. Infurther embodiments of the present invention, divalent cations can beincluded in an amount sufficient to displace or block the effect ofcalcium ions at the cellular membrane. Accordingly, in some embodiments,Ca⁺⁺ is provided in a range of from about 0.01 mM to 0.1 mM, preferablyfrom about 0.01 to 0.07 mM, and Mg⁺⁺ is provided in a range of fromabout 1 mM to 10 mM, preferably about 2.5 mM to 7.5 mM.

2) Oncotic Agents

In some embodiments of the present invention, the media comprises one ormore oncotic agents. In preferred embodiments, the oncotic agent isincluded in an amount sufficient to maintain oncotic pressure equivalentto that of blood plasma. The present invention is not limited to anyparticular oncotic agent. Indeed, any oncotic agent can be used that isof a size that does not readily escape the circulation by traversing thefenestrations of the capillary bed. Examples of oncotic agents include,but are not limited to, hydroxyethyl starch, cyclodextrins, and dextran(e.g., Dextran 30, 40, or 50). In preferred embodiments, the mediacomprises from about 1% to 10% of the oncotic agent. In particularlypreferred embodiments, the media comprises about 5% of the oncoticagent. Surprisingly, it has been found that the hydroxyethyl starchcomponent of VIASPAN can be deleted and good results still obtained.

3) Buffers

In some embodiments of the present invention, the media comprises atleast one buffer. In preferred embodiments, the concentration ofbuffer(s) is sufficient to maintain the pH of the media at a range offrom about 7.0 to 8.0 at 10° C., preferably from about 7.4 to 7.8. Thepresent invention is not limited to the use of any particular buffer.Indeed, the use of a variety of synthetic and other buffers iscontemplated. Examples of suitable buffers include, but are not limitedto, N-2-hydroxyethylpiperazine-N′-2-hydroxypropanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid;2-((2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino)ethanesulfonic acid(TES), 3-(N-tris(hydroxy-methyl)methylamino)-2-hydroxypropanesulfonicacid (TAPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid(EPPS), pH range 7.3-8.7, and tris(hydroxymethyl)aminomethane (THAM),HCO₃, and H₂PO₄.

4) Energy Sources

In some embodiments of the present invention, the media furthercomprises one or more energy or nutrition sources. Examples of energysources include, but are not limited to, sucrose, fructose, glucose, anddextran. Preferably, the concentration of the energy source is fromabout 1 mM to 20 mM, most preferably about 10 mM.

5) Impermeant Anions

In some embodiments of the present invention, the media comprises one ormore impermeant anions. The impermeant anion is included to counteractswelling during cold exposure. The present invention is not limited toany particular impermeant anion. Indeed, a variety of impermeant anionsare contemplated, including, but not limited to, gluconate andlactobionate. Preferably, the concentration of the impermeant anion isfrom about 50 to 150 mM, most preferably about 100 mM.

6) Free Radical Scavengers

In some embodiments of the present invention, the media comprises a freeradical scavenger. The present invention is not limited to anyparticular free radical scavenger. Indeed, a variety of free radicalscavengers are contemplated including, but not limited to, mannitol andglutathione. Preferably, the concentration of the free radical scavengeris from about 1 mM to 10 mM, most preferably about 3 mM.

7) ATP Substrate

In some embodiments of the present invention, the media comprises one ormore ATP substrates for the regeneration of ATP during rewarming. Thepresent invention is not limited to any particular ATP substrate.Indeed, a variety of ATP substrates are contemplated, including, but notlimited to, adenosine, fructose, adenine, and ribose. Preferably, theconcentration of the ATP substrate is from about 1 mM to 10 mM, mostpreferably about 5 mM.

8) Osmotic Agents

In some embodiments of the present invention, the media comprises one ormore osmotic agents. Examples of osmotic agents include, but are notlimited to, trehalose (α-α-trehalose dihydrate), raffinose, sucrose andmannitol. In preferred embodiments, the osmotic agent is provided at aconcentration of about 1 mM to 100 mM, most preferably about 30 mM. Inother embodiments, it is contemplated that trehalose is included in themedia as protectant. Accordingly, in some embodiments, the mediacomprises trehalose at a concentration of about 1 mM to 30 mM,preferably about 20 mM. In other embodiments, trehalose is included inthe media at a concentration sufficient to lower serum creatinine levelsin kidney transplant recipients as compared to recipients of kidneysstored without antimicrobial peptides.

9) Other Components

In some embodiments of the present invention, the media may furthercomprise a variety of additional components. For example, in someembodiments, the media comprises an inhibitor of xanthine oxidase (e.g.,allopurinol at a concentration of about 0.1 mM to 5 mM, most preferablyabout 1 mM). In still further embodiments, the media comprises aniron-chelating agent (e.g., deferoxamine at a concentration of fromabout 0.05 mM to 5 mM, most preferably about 1.0 mM). In additionalembodiments, the media comprises a steroidal agent (e.g., dexamethasoneat a concentration of about 1 to 30 mg/liter, most preferably about 16mg/liter). In other embodiments, the media comprises hydrocortisone(e.g., at a concentration of from about 10 ng/ml to 100 ng/ml,preferably about 36 ng/ml). In still other embodiments, the mediacomprises ITS (insulin [5 μg/ml], transferrin [5 μg/ml], and selenium [5ng/ml]). In some embodiments, the media comprises vitamin C (e.g., atabout 1×10⁻⁷ M). In other embodiments, the media comprises proteaseinhibitors (e.g., phosphoramidon [5 μM], thiorphan [1 μM], bacetracin [1μM], and encaptopril [5 μM]).

Additionally, the media of the present invention may comprise additionalcytoskeleton stabilizing agents. In particular, agents such as taxol,discodermolide, epothilone A and B, vinblastine, and vinchristine may beutilized in some embodiments of the present invention, in combinationwith either the antimicrobial polypeptides or cell surface receptorbinding compounds or both. The use of taxol with UW solution isdescribed in U.S. Pat. No. 5,696,152, incorporated herein by reference.

10) Exemplary Media Formulations

It is contemplated that antimicrobial peptides, other pore formingagents, and/or cell surface receptor binding compounds can be added to avariety of media formulations currently being used for organpreservation and/or other surgical procedures such as cardioplegia.Non-limiting examples of these media are provided in the Tables below.It will be recognized that the media may comprise one or moreantimicrobial polypeptides (e.g., a defensin such as BNP-1). The mediadescribed below may also comprise one or more trophic factors and/orcell surface receptor binding compounds as described above. Accordingly,in some preferred embodiments, the media is supplemented with one ormore of the following trophic factors: trehalose (Sigma, St. Louis Mo.;e.g., about 15 mM), substance P (Sigma; e.g., about 10 μg/ml), IGF-1(Collaborative Biologicals; e.g., about 10 ng/ml), EGF (Sigma; e.g.,about 10 ng/ml), and NGF (Sigma [murine] or Genentech [human]; e.g.,about 200 ng/ml). In some preferred embodiments, the transplant media isalso supplemented with dexamethasone (1-20 mg/l, preferably 8 mg/l),penicillin (20,000-500,000 units, preferably 200,000 units), and insulin(1-200 units, preferably 40 units) prior to use. In some embodiments, anantimicrobial polypeptide is not included in the medium. In someembodiments, the antimicrobial polypeptide and/or cell surface receptorbinding compounds are included in the media at concentrations sufficientto lower serum creatinine levels in kidney transplant recipients ascompared to recipients of kidneys stored in control unsupplementedmedia. In other embodiments, the antimicrobial polypeptide and/or cellsurface receptor binding compounds are included in the media atconcentrations sufficient to cause a decrease in delayed graft functionrates of kidneys stored in the media as compared to controlunsupplemented media. Preferably, the time for return to baseline serumcreatinine levels is improved by at least 25%, and most preferably by atleast 50%, as compared to control unsupplemented media.

It is contemplated that the media can be provided in a pre-formulatedform (which can be in kit format with instructions, etc.) whichcomprises the antimicrobial polypeptide and/or one or more trophicfactors or as a kit comprising at least one container of base medium(e.g., UW solution (VIASPAN), HTK Solution, EuroCollins Solution, orCollins Solution)) and a separate container or containers containing atleast one of the antimicrobial polypeptides and/or one or more cellsurface receptor binding compounds. Therefore, it will be recognizedthat the Tables below provide formulations for exemplary supplementedmedia (i.e., the formula of the media after addition of theantimicrobial polypeptide and at least one cell surface receptor bindingcompound) and that the media can be provided in either a pre-formulatedform or supplemented immediately prior to use. In preferred embodiments,the antimicrobial polypeptide and/or one or more cell surface receptorbinding compounds are provided in stable form that can be reconstituted.Methods for stabilization include lyophilization. In embodiments wherethe antimicrobial polypeptide and/or one or more cell surface receptorbinding compounds are provided in lyophilized form, they canconveniently reconstituted prior to use in sterile water or in analiquot of base medium (e.g., UW solution) prior to addition to the basemedium (e.g., UW solution). In some embodiments, the kits includeinstructions for reconstitution of the antimicrobial polypeptide and/orone or more cell surface receptor binding compounds and/or for the useof the supplemented medium for cold storage or machine perfusion of anorgan.

Alternatively, the at least one microbial polypeptide and/or one or morecell surface receptor binding compounds can be provided as a separatecomposition (i.e., a “bullet”) that is added to a base medium. Inpreferred embodiments, the bullet contains a defensin and/or one or moreof the cell surface receptor binding compounds described above. In someembodiments, the bullet contains a defensin and/or one or more of thecell surface receptor binding compounds above in concentrations thatprovide the appropriate concentration when added to one liter, twoliters, or five liters of the base medium. For example, in somepreferred embodiments, a bullet for addition to 1 liter of base mediumcomprises 1 mg of an antimicrobial polypeptide (e.g., BNP-1), 10 mgSubstance P, 10 μg IGF-1, 10 μg EGF, 200 μg NGF, and an amount oftrehalose sufficient to provide a concentration of 15 mM. In otherpreferred embodiments, a bullet for addition to 1 liter of base mediumcomprises 1 mg of an antimicrobial polypeptide (e.g., BNP-1), 10 mgSubstance P, 10 μg IGF-1, and 10 μg EGF. In still other preferredembodiments, the antimicrobial polypeptide and/or one or more cellsurface receptor binding compounds are provided in amounts such when thebullet is added to a base transplant medium and the supplemented mediumis used for kidney storage prior to transplantation, subjects receivingthe kidneys stored in the supplemented medium exhibit a faster return tobaseline serum creatinine levels than patients receiving kidneys storedin unsupplemented medium.

TABLE 3 Supplemented UW Solution (VIASPAN) Lactobionic acid 100 mM KOH100 mM NaOH 20 mM Adenosine 5 mM Allopurinol 1 mM Potassium Phosphate 25mM (Monobasic) MgSO₄ 5 mM Raffinose 30 mM Glutathione 3 mM Hydroxyethylstarch 50 g/L Defensin 1 mg/L dexamethasone 8 mg/l penicillin 200,000units insulin 40 units pH 7.4

TABLE 4 Supplemented UW Machine Perfusion Solution Hydroxyethyl starch50 g/L Potassium gluconate 10 mM Sodium gluconate 90 mM PotassiumPhosphate (Monobasic) 15 mM Glucose 10 mM Glutathione 3 mM HEPES 10 mMMagnesium gluconate 5 mM Calcium chloride 0.5 mM Ribose 5 mM Adenosine 5mM Adenine 5 mM Allopurinol 1 mM Mannitol 14 mM Defensin 1 mg/L pH 7.4Osmolarity 310

TABLE 5 Hypertonic Citrate Solution Na⁺ 80 mM K⁺ 80 mM Mg⁺⁺ 35 mMCitrate⁻ 55 mM SO₄ ⁻ 35 mM Mannitol 136 mM Defensin 1 mg/L pH 7.1Osmolarity 400

TABLE 6 HTK Solution Na⁺ 15 mM K⁺ 10 mM Mg⁺⁺ 4 mM Cb⁻ 50 mM Tryptophan 2mM 2-oxoglutarate 1 mM Mannitol 30 mM Histidine 0.18 mM Histidine HCl 18mM pH 7.3 Defensin 1 mg/L Osmolarity 310

TABLE 7 HTK Solution of Bretschneider Ketoglutaric acid 1 mM Tryptophan2 mM MgCl₂ 4 mM KCl 10 mM NaCl 15 mM Histidine 200 mM Defensin 1 mg/L pH7.3

TABLE 8 Phosphate Buffered Sucrose Sodium Phosphate 53.6 mM DibasicSodium Phosphate 15.5 mM Monobasic Sucrose 140 mM Defensin 1 mg/L pH 7.2

TABLE 9 EuroCollins Solution NaHCO₃ 10 mM KCl 15 mM K₂HPO₄ 42.5 mMKH₂PO₄ 15.1 mM Glucose 195 mM Defensin 1 mg/L

TABLE 10 Collins C2 Solution K₂HPO₄ 42.5 mM KH₂PO₄ 15.1 mM KCl 15 mMNaHCO₃ 10 mM Glucose 140 mM MgSO₄ 30 mM Defensin 1 mg/L

TABLE 11 Supplemented UW Solution (VIASPAN) Lactobionic acid 100 mM(potassium lactobionate) KOH 100 mM NaOH 20 mM Adenosine 5 mMAllopurinol 1 mM Potassium Phosphate (Monobasic) 25 mM MgSO₄ 5 mMRaffinose 30 mM Glutathione 3 mM Hydroxyethyl starch 50 g/L BNP-1 1 mg/LTrehalose 15 mM Substance P 10 μg/ml IGF-1 10 ng/ml EGF 10 ng/ml NGF 200ng/ml dexamethasone 8 mg/l penicillin 200,000 units insulin 40 units pH7.4

TABLE 12 Supplemented UW Solution (VIASPAN) Lactobionic acid 100 mM(potassium lactobionate) KOH 100 mM NaOH 20 mM Adenosine 5 mMAllopurinol 1 mM Potassium Phosphate (Monobasic) 25 mM MgSO₄ 5 mMRaffinose 30 mM Glutathione 3 mM Hydroxyethyl starch 50 g/L BNP-1 1 mg/LSubstance P 10 μg/ml IGF-1 10 ng/ml EGF 10 ng/ml dexamethasone 8 mg/lpenicillin 200,000 units insulin 40 units pH 7.4

TABLE 13 EuroCollins Solution NaHCO₃ 10 mM KCl 15 mM K₂HPO₄ 42.5 mMKH₂PO₄ 15.1 mM Glucose 195 mM Trehalose 15 mM Substance P 10 μg/ml IGF-110 ng/ml EGF 10 ng/ml NGF 200 ng/ml BNP-1 1 mg/L EuroCollins SolutionNaHCO₃ 10 mM KCl 15 mM K₂HPO₄ 42.5 mM KH₂PO₄ 15.1 mM Glucose 195 mMSubstance P 10 μg/ml IGF-1 10 ng/ml EGF 10 ng/ml BNP-1 1 mg/L

TABLE 14 Supplemented UW Solution (VIASPAN) Lactobionic acid 100 mM(potassium lactobionate) KOH 100 mM NaOH 20 mM Adenosine 5 mMAllopurinol 1 mM Potassium Phosphate (Monobasic) 25 mM MgSO₄ 5 mMRaffinose 30 mM Glutathione 3 mM Hydroxyethyl starch 50 g/L Trehalose 15mM Substance P 10 μg/ml IGF-1 10 ng/ml EGF 10 ng/ml NGF 200 ng/mldexamethasone 8 mg/l penicillin 200,000 units insulin 40 units pH 7.4

TABLE 15 Supplemented UW Solution (VIASPAN) Lactobionic acid 100 mM(potassium lactobionate) KOH 100 mM NaOH 20 mM Adenosine 5 mMAllopurinol 1 mM Potassium Phosphate (Monobasic) 25 mM MgSO₄ 5 mMRaffinose 30 mM Glutathione 3 mM Hydroxyethyl starch 50 g/L Substance P10 μg/ml IGF-1 10 ng/ml EGF 10 ng/ml dexamethasone 8 mg/l penicillin200,000 units insulin 40 units pH 7.4

TABLE 16 EuroCollins Solution NaHCO₃ 10 mM KCl 15 mM K₂HPO₄ 42.5 mMKH₂PO₄ 15.1 mM Glucose 195 mM Trehalose 15 mM Substance P 10 μg/ml IGF-110 ng/ml EGF 10 ng/ml NGF 200 ng/ml

TABLE 17 EuroCollins Solution NaHCO₃ 10 mM KCl 15 mM K₂HPO₄ 42.5 mMKH₂PO₄ 15.1 mM Glucose 195 mM Substance P 10 μg/ml IGF-1 10 ng/ml EGF 10ng/mlII. Uses of Media

It is contemplated that the media described above may be utilized in avariety of transplant and other medical procedures. It is contemplatedthat the media can be used for the preservation of any tissue, organ,cell(s), or organisms, including, but not limited to, organs,genetically engineered tissues, biomedically engineered tissues, sperm,eggs, and embryos. In particular, the media finds use for thepreservation of both internal and external organs prior to transplant.In some embodiments, the media is utilized for hypothermic storage ofthe organ. In hypothermic storage, the organ is flushed with the media,cooled, suspended in the media, and stored. In other embodiments, themedia is utilized for pulsatile hypothermic perfusion of the organ. Instill further embodiments, the present invention provides a compositioncomprising an internal organ suspended in or perfused with a mediacomprising one or more antimicrobial polypeptides (e.g., defensins)and/or at least one cell surface receptor binding protein. Inparticularly preferred embodiments, the media of the present inventionare useful for decreasing the incidence and/or severity of delayed graftfunction in patients receiving transplanted kidneys stored and/ortreated with the media of the present invention.

In other embodiments, the present invention provides a compositioncomprising skin or another external organ suspended in or perfused witha media comprising an antimicrobial peptide or other pore forming agentsand/or at least one growth factor. In other embodiments, the media maybe used in procedures such as cardioplegia (See, e.g., U.S. Pat. No.5,514,536, incorporated herein by reference).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

Example 1

This Example describes the use of media comprising defensins for thestorage of organs prior to transplant. The study was performed on adultbeagle dogs of both sexes weighing approximately 8 kg. The studyemployed a kidney autotransplantation with immediate contralateralnephrectomy model. This involved harvesting of either the left or rightkidney and flushing it out through the renal artery with the Universityof Wisconsin solution (See Table 3) either with or without addeddefensins (1 mg/liter), storage of the kidney under sterile conditionson ice for 3 days, reimplantation of the previously harvested kidneyinto the abdominal cavity of the same dog and then immediately removingthe other kidney.

For harvest of the kidney, a midline abdominal incision was made and theleft kidney isolated by dissecting free of any attachments to itsartery, vein and ureter. The ureter was ligated with a single 4-0 silkligature near the bladder and divided proximal to the ligature. Thegonadal vein was ligated with 2 4-0 silk ligatures and divided. Therenal artery and vein were then clamped and cut and the kidney removedfor vascular flushing with preservation solution and experimentalstorage. The kidney was then suspended in preservation solution insterile plastic bags and placed on ice in a cooler for storage. Thestumps of the renal artery and vein were ligated separately with doubled3-0 silk ligatures. The excision site was inspected for hemorrhage andany small bleeders were cauterized or ligated. The body wall was closedwith O-Maxon in a simple continuous pattern. The skin was then closedwith 3-0 Vicryl in a simple continuous subcuticular pattern after whichthe dog was recovered from anesthesia.

Three days after harvest of the kidney the dog was anesthetized forreimplantation of the stored kidney. Intravenous morphine (0.5 mg/kg)was administered as prophylaxis against intussusception. The abdomen wasentered through a midline abdominal incision made by opening theprevious incision and extending the incision to the pubis. The externaliliac artery and common iliac vein were isolated by blunt and sharpdissection. The external iliac artery was ligated distally, clampedproximally with an atraumatic vascular clamp and divided just proximalto the ligature. The free arterial end was flushed with heparinizedsaline and its end cleared of loose adventitia. The common iliac veinsurface was cleared of loose adventitia by sharp dissection. Anatraumatic vascular clamp was placed on the vein both proximally anddistally and the vein wall fenestrated using a Metzenbaum scissors. Thevein segment was flushed free of blood with heparinized saline. Four 7-0polyester sutures were placed in the wall of the vein exiting thefenestration and attached to the renal vein. The renal vein was apposedto the side of the iliac vein and the anastomosis performed using twolines (front and back vessel walls) of simple continuous suture. Therenal artery was apposed to the end of the external iliac artery usingtwo 7-0 polypropylene sutures and the anastomosis completed with twolines of simple continuous suture. The proximal venous clamp was removedfollowed by the arterial vascular clamp. Mannitol (1 gm/kg IV) wasadministered during anastomosis, which required less than 30 minutes tocomplete. The bladder was entered through a ventral incision andfenestrated on its dorsal side with a hemostat. The ureter was incisedlongitudinally for 1 cm and then pulled through the bladderfenestration. The ureteral mucosa and bladder mucosa was apposed using6-0 Vicryl suture in a continuous pattern. The bladder was closed with3-0 Vicryl in a Cushing pattern. The contralateral kidney was excisedand the ureter, renal artery, and renal vein ligated with 3-0 silk. Theabdominal wall was closed with O-Maxon and the skin with 3-0 Vicrylusing continuous suture patterns in the linea alba and subcuticularlayers, respectively. The dog was then given 500 ml lactated Ringer'ssolution subcutaneously and recovered from anesthesia.

The results are presented in FIG. 1. As can be seen, dogs receivingkidneys stored for three days in UW solution supplemented with BNP-1exhibited serum creatinine of about half that seen in dogs receivingkidneys stored in UW solution alone. This is indicative of markedlyimproved renal function in kidneys preserved in media containing BNP-1.

Example 2

This Example describes the use of media comprising defensins and/or cellsurface receptor binding compounds for the storage of organs prior totransplant. The study was performed as described in Example 1, exceptthat the organs were stored for four days prior to transplant. The threetest groups were UW solution alone, UW solution supplemented with 1 mg/LBNP-1 (synthesized by Multiple Peptide Systems, San Diego Calif.), andUW solution supplemented with 1 mg/L BNP-1, and the following trophicfactors: 20 mM trehalose (Sigma, St. Louis Mo.), 2.5 mg/L substance P(Sigma), 10 μg/L IGF-1 (Collaborative Biologicals), 10 μg/L EGF (Sigma),and 200 ng/ml NGF (Sigma [murine] or Genentech [human])). The resultsare presented in FIG. 2. As can be seen, dogs receiving kidneys storedin UW solution supplemented with BNP-1 and cell surface receptor bindingcompounds exhibited serum creatinine of about half that seen in dogsreceiving kidneys stored in UW solution supplemented with BNP-1 or UWsolution alone. Surprisingly, the serum creatinine levels in the dogsreceiving kidneys stored in UW solution supplemented with both BNP-1 andcell surface receptor binding compounds remarkably improved the qualityof preservation to the point that they equal 3 day BNP-1 preservedkidneys and 2 day or less storage with UW solution alone.

Example 3

This Example describes results from the transplant of kidneys after sixdays of storage. This study was performed as described in Example 1,except that the kidneys were stored for four days in UW solution priorto transplant or six days in UW solution supplemented with a defensinand trophic factors (See Example 2) prior to transplant. The results arepresented in FIG. 3. As can be seen, the serum creatinine levelsfollowing transplant were similar in the two groups. These datademonstrate that UW solution supplemented with trophic factors can beused increase the duration of storage.

Example 4

This Example describes results from the transplant of kidneys after sixdays of storage. This study was performed as described in Example 3,except that the kidneys were stored for three days in UW solution priorto transplant or six days in UW solution supplemented with a defensinand trophic factors (See Example 2) prior to transplant. The results arepresented in FIG. 4. As can be seen, the serum creatinine levelsfollowing transplant were higher in the dogs receiving kidneys storedfor six days as opposed dogs receiving kidneys stored for three days.These data demonstrate that UW solution supplemented with trophicfactors can be used increase the duration of storage.

Example 5

This Example describes the results from the transplant of kidneys afterfive days of storage. This study was performed as described in Example3, except that the kidneys were stored for three days in UW solutionprior to transplant or five days in UW solution supplemented with adefensin and trophic factors (See Example 2) prior to transplant. Theresults are presented in FIG. 5. As can be seen, the serum creatininelevels following transplant were higher in the dogs receiving kidneysstored for three days in UW solution as opposed dogs receiving kidneysstored for five days in UW solution plus trophic factors. These datademonstrate that UW solution supplemented with trophic factors can beused increase the duration of storage.

Example 6

This Example describes the results from the transplant of kidneys afterfour days of storage. This study was performed as described in Example3, except that the kidneys were stored for three days in UW solutionprior to transplant or four days in UW solution supplemented with adefensin and trophic factors (See Example 2) prior to transplant. Theresults are presented in FIG. 6. As can be seen, the serum creatininelevels following transplant were significantly higher in the dogsreceiving kidneys stored for three days in UW solution as opposed dogsreceiving kidneys stored for four days in UW solution plus trophicfactors. These data demonstrate that UW solution supplemented withtrophic factors can be used increase the duration of storage areindicative of markedly improved renal function in kidneys preserved inmedia containing trophic factors.

Example 7

This Example describes the results from the transplant of kidneys afterfour days of storage. This study was performed as described in Example3, except that the kidneys were stored for four days in UW solutionprior to transplant or four days in UW solution supplemented with adefensin and trophic factors (See Example 2) prior to transplant. Theresults are presented in FIG. 7. As can be seen, the serum creatininelevels following transplant were significantly higher in the dogsreceiving kidneys stored for four days in UW solution as opposed dogsreceiving kidneys stored for four days in UW solution plus trophicfactors. These data are indicative of markedly improved renal functionin kidneys preserved in media containing trophic factors.

Example 8

This Example demonstrates that hydroxyethyl starch can be deleted fromUW solution without adversely affecting organ quality. This study wasperformed as described in Example 3, except that the kidneys were storedfor five days prior to transplant in UW solution containing hydroxyethylstarch and supplemented with trophic factors or five days prior totransplant in UW solution supplemented with trophic factors (See Example2), and in which the hydroxyethyl starch was omitted. The results arepresented in FIG. 8. Surprisingly, the serum creatinine levels followingtransplant were significantly higher in the dogs receiving kidneysstored in UW solution containing hydroxyethyl starch as opposed dogsreceiving kidneys stored in UW solution without hydroxyethyl starch.

Example 9

This Example demonstrates experiments where use of the D-form isomer ofBNP-1 was compared with L-form isomer. The D-form isomers wassynthesized with D-amino acids. This study was performed as described inExample 1, except that the kidneys were stored for three days prior totransplant in UW solution containing the L-form isomer of BNP-1 or threedays prior to transplant in UW solution containing the D-form isomer ofBNP-1. The results are presented in FIG. 9. As can be seen, dogsreceiving kidneys stored in media supplemented with the D-form isomerreturned to normal serum creatinine levels faster than dogs receivingkidneys stored in the media supplemented with the L-form isomer.

Example 10

This Example describes the effect UW solution supplemented with BNP-1 oncytoskeletal structure of kidney cells. Briefly, either MDCK cells orprimary kidney cell cultures were stored for three days at coldtemperatures in either UW solution, UW solution supplemented with BNP-1,or DMEM. The cells were then labeled with actin and tubulin antibodiesand analyzed by confocal fluorescence microscopy. Control untreatedcells displayed a homogeneous fine fibrillar pattern of actin andtubulin that extended throughout the cell. Cells stored in DMEM culturemedia at cold temperatures displayed nearly complete dissolution of bothactin and tubulin with very little staining present. Cells stored in UWsolution had nearly complete disruption of the tubulin elements andsignificant dissolution of the actin microfilaments. In primary culturesin UW solution, the residual actin in condensed along the plasmamembrane. Treatment with BNP-1 during storage resulted in bettermaintenance of actin and tubulin in MDCK cells. In primary cultures withBNP-1, the tubulin and actin were better stained and more persistentwith some condensation along stellate rays which extended from thenucleus out to the plasma membrane of the cells In a separateexperiment, the effect of BNP-1 on the cytoskeleton after three dayscold storage in UW solution followed by 3 hours warm reperfusion in DMEMculture media with 10% serum was determined. MDCK cells stored in DMEMculture media at 4° C. failed to reassemble the cytoskeleton by 3 hoursof reperfusion. MDCK cells stored in UW solution and then reperfusedwere able to reassemble the cytoskeleton, but in primary kidney cellcultures the cytoskeleton remained abnormal at 3 hours of reperfusion.In these primary cells, the actin and tubulin filaments maintained acoarse clumpy pattern with considerable cortical condensation near theplasma membrane and only a limited amount of fine fibrillar structurethat would be considered more normal. Cells stored in BNP-1 supplementedUW solution and reperfused had superior maintenance and reassembly ofthe cytoskeleton in both MDCK and primary renal cultures withhomogeneously distributed fine fibrillar cytoskeletal elementspredominating in these cells.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in organstorgae and transplant, cryobiology, biochemistry, or related fields areintended to be within the scope of the following claims.

1. An in vitro composition comprising an internal organ at a temperatureof 0-4° C., lactobionate, Substance P, nerve growth factor, andrecombinant insulin-like growth factor 1, said recombinant Insulin-likeGrowth Factor 1 provided at a concentration of from about 1 ng/ml to 100ng/ml.
 2. The composition of claim 1, further comprising hydroxyethylstarch.
 3. The composition of claim 2, wherein said hydroxyethyl starchis present in a concentration of about 1 g/l to 200 g/l.
 4. Thecomposition of claim 1, further comprising an antimicrobial polypeptide.5. The composition of claim 4, wherein said antimicrobial polypeptide isa defensin.
 6. The composition of claim 5, wherein the amino acidsequence for said defensin is SEQ ID NO:
 37. 7. The composition of claim4, wherein said antimicrobial polypeptide is present in a concentrationof about 0.01 mg/l to 1000 mg/l.
 8. The composition of claim 1, whereinsaid lactobionate is present in a concentration of about 1 mM to 500 mM.9. The composition of claim 1, wherein said Substance P is provided at aconcentration of from about 0.1 μg/ml to 100 μg/ml.
 10. The compositionof claim 1, wherein said Nerve Growth Factor is provided at aconcentration of from about 1 ng/ml to 100 ng/ml.
 11. An in vitrocomposition comprising an internal organ at a temperature of 0-4° C.,lactobionate at a concentration of about 1 mM to 500 mM, Substance P.nerve growth factor, and recombinant insulin-like growth factor 1 at aconcentration of from about 1 ng/ml to 100 ng/ml.
 12. An in vitrocomposition comprising an internal organ, lactobionate at aconcentration of about 1 mM to 500 mM, recombinant insulin-like growthfactor 1 at a concentration of from about 1 ng/ml to 100 ng/ml,hydroxyethyl starch at a concentration of about 1 g/l to 200 g/l, NerveGrowth Factor at a concentration of from about 1 ng/ml to 100 ng/ml, andSubstance P at a concentration of from about 0.1 μg/ml to 100 μg/ml.